Nerve regeneration promoters containing semaphorin inhibitor as the active ingredient

ABSTRACT

To provide a semaphorin inhibitor; a peripheral or central nerve regeneration promoter which contains said semaphorin inhibitor as an active ingredient; and a preventive or remedy for a neuropathic disease and a neurodegenerative disease containing said nerve regeneration promoter, or the like.  
     A low-molecular weight compound, which acts at a concentration of 10 μg/ml or below to inhibit the growth cone collapse activity of semaphorin such as semaphorin 3A, semaphorin 6C or the like and/or the nerve outgrowth inhibitory activity of semaphorin in a collagen gel and which does not substantially affect cell proliferation, is obtained from the culture of strain SPF-3059 belonging to the genus Penicillium. The low-molecular weight compound with the semaphorin inhibitory activity thus obtained exhibits the in vivo nerve-regeneration promoting action.

TECHNICAL FIELD

[0001] The present invention relates to central or peripheral nerveregeneration promoters containing semaphorin inhibitor as an activeingredient, to preventives and remedies for neurodegenerative diseasesor the like which contain the nerve regeneration promoters, tonon-peptide and non-nucleotide semaphorin inhibitors, to compounds withthe semaphorin inhibitory activity, and to a microbiological process forproducing the said compounds or the like.

BACKGROUND ART

[0002] Nerve cells are particular tissues, which has no mitoticpotential in an adult. Therefore, once they are injured, the damage willlast over a long period of time. It is said that there is noregeneration potential especially in the central nervous system (CNS)such as brain and spinal cord. Lack of the regeneration potential in thecentral nerves can be regarded as one of the reasons that there havebeen no established therapies for traumatic injuries such as spinal cordinjury, nor for neurodegenerative diseases such as Alzheimer's diseaseand Parkinson disease. On the other hand, peripheral nerves possessregeneration potential. Their axons can regenerate and their functionscan be recovered even after having been severed. In this case, however,the recovery requires a long span of time ranging from several months toeven more than a year, and thus patients have to undergo considerablesufferings. Moreover, the recovery period is so long that some nervecells may die during this period, which often leads to the failure ofrecovery of the functions. And yet, even the peripheral nerves havingregeneration potential are entirely unable to outgrow in the CNS such asin brain and spinal cord. This brings the basis for the hypothesis thatthere exist some substances in the central nervous system that inhibitnerve outgrowth. If the inhibitory substances for nerve regeneration inthe central nervous system are suppressed by using antibodies or thelike, nerve regeneration in the CNS as well as the recovery of theirfunctions will be observed, even though partially. As one of suchinhibitory substances for the central nerve regeneration, Nogo has beenrecently discovered (Nature 403, 434, 2000, Nature 403, 439, 2000).However, only a small portion of axons are regenerated by inhibitingNogo and it is thus presumed that there exist some otherregeneration-inhibitory substances. The followings are exemplified ascandidate substances that inhibit the nerve regeneration: MAG(myelin-associated glycoprotein); tenascin; gangliosides; ephrin;netrin; slit; semaphorins and the like. The only reason for thesesubstances to be exemplified as the candidate substances is that theyinhibit neurite outgrowth in vitro. Whether they actually act to inhibitnerve regeneration in vivo has not yet been elucidated so far.

[0003] Referring to semaphorin, its gene was first isolated as a factorinvolved in nervous system formation in developing locusts. Since then,it has been reported that semaphorins constitute a large gene familydistributing in nematodes, fish, mammals or even certain kinds of virus,and currently semaphorin genes are classified into eight genesubfamilies or classes based on their structures (Cell 97, 551, 1999).Semaphorin is an endogenous protein identified as a factor whichcollapses nerve growth cone and suppresses axon outgrowth, and so far,about 20 molecular species have been reported (Cell 97, 551, 1999).However, most functions of many semaphorin families have not yet beendiscovered in detail. The most studied gene group is that of a subfamilycalled the class III type, all of whose translation products aresecretory proteins. Although proteins encoded by these genes are knownto possess intensive neurite outgrowth suppressing activity and growthcone collapse activity in vitro, it was reported that they induceneurite outgrowth under certain conditions. Among them, semaphorin 3A(Sema3A) is the most studied and is known to induce growth cone collapseof the cultured nerve cells at as low as 10 pM concentration in a shortperiod of time (Cell 75, 217, 1993, Cell 75, 1389, 1993). In order toanalyze in vivo functions of semaphorins, knockout mice forneuropilin-1, which is one of the components of Sema3A receptor, havebeen studied (Neuron 19, 995, 1997). The said knockout mice showembryonic lethality as well as motor abnormality in some nervous systemssuch as trigeminal nerve and angiogenesis abnormality. Although similarmotor abnormality in nervous systems is observed in Sema3A knockoutmice, some individual mice are reported to grow up to adults withoutserious problem. Therefore, the in vivo functions of Sema3A remainlargely unknown.

[0004] Moreover, with regard to semaphorins the followings are alsoknown: antisense nucleototides and antagonists such as antibodies or thelike for semaphorin W, semaphorin Y and semaphorin Z are made forcentral nerve regeneration promoters (WO98/15628, WO98/11216,WO98/20928); and a method of inducing neurite outgrowth by contacting anerve cell with an antibody that specifically binds to human collapsinis known (U.S. Pat. No. 5,416,197). However, in vivo actions of theseantibodies or the like still remain unknown. Besides, any low-molecularweight compounds other than peptide or nucleotide such as an antibody oran antisense nucleotide or the like, which specifically inhibitssemaphorin have been completely unknown.

[0005] The subject of the present invention is to provide semaphorininhibitors, peripheral or central nerve regeneration promoters thatcontain the said semaphorin inhibitors as an active ingredient,preventives and remedies for neuropathic diseases and neurodegenerativediseases that contain the nerve regeneration promoters.

DISCLOSURE OF THE INVENTION

[0006] Semaphorins are thought to have various actions, and someresearchers postulated that semaphorins were involved not only in thenerve development but also in the nerve regeneration, though there werelittle evidence. Further, a gene deficient animal does not always serveas a tool for evaluating the function of the gene because the loss ofthe gene function often induce a compensatory action by other genes, asobserved in MAG knockout animal. Although MAG had been thought for yearsas a result of a number of studies to be one of inhibitory substancesfor nerve regeneration, MAG knockout mice did not show promotion ofnerve regeneration. The present inventors, therefore, commenced thestudy on semaphorin to examine whether inhibition of semaphorin bringsabout the in vivo nerve regeneration without using semaphorin knockoutanimals.

[0007] First, a substance that entirely inhibits the semaphorin activityin vitro was screened, and thus screened semaphorin inhibitor wasadministered to a nerve regeneration animal model to clarify the in vivonerve-regeneration promoting activity of said semaphorin inhibitor. As aresult of this study, the present inventors have found that a substance,which not only inhibits the growth cone collapse activity of semaphorinentirely but also persistently inhibits the semaphorin activity in acollagen gel and promotes neurite outgrowth in the presence ofsemaphorin, and exhibits the in vivo nerve-regeneration promotingactivity. The present inventors have further found that nerveregeneration was promoted not only in the peripheral nervous system butin the CNS by using the semaphorin inhibitor. The study not onlyelucidated the in vivo action of semaphorin but also made it possible toprovide nerve regeneration promoter containing a semaphorin inhibitor,which has been completely undiscovered, as an active ingredient, and apharmaceutical containing the nerve regeneration promoter. The studyhave also elucidated that a compound with a specific structure in themolecule discovered in the culture of Penicillium sp. SPF-3059, whichwas screened by the present inventors, exhibits semaphorin inhibitoryactivity. The present invention was accomplished based on thesefindings.

[0008] The present invention relates to: a nerve regeneration promotercontaining an inhibitor for a nerve outgrowth repelling factor as anactive ingredient (claim 1); the nerve regeneration promoter accordingto claim 1, wherein the inhibitor for a nerve outgrowth repelling factoris a semaphorin inhibitor (claim 2); the nerve regeneration promoteraccording to claim 2, wherein the semaphorin inhibitor is a class 3semaphorin inhibitor (claim 3); the nerve regeneration promoteraccording to claim 3, wherein the class 3 semaphorin inhibitor is asemaphorin 3A inhibitor (claim 4); the nerve regeneration promoteraccording to claim 2, wherein the semaphorin inhibitor is a class 6semaphorin inhibitor (claim 5); the nerve regeneration promoteraccording to claim 5, wherein the class 6 semaphorin inhibitor is asemaphorin 6C inhibitor (claim 6); the nerve regeneration promoteraccording to any of claims 2-6, wherein the semaphorin inhibitor is acompound having promoting action on the central and/or the peripheralnerve regeneration (claim 7); the nerve regeneration promoter accordingto any of claims 2-7, wherein the semaphorin inhibitor is a compoundhaving suppressing action on the growth cone collapse activity ofsemaphorin and/or on the nerve outgrowth inhibitory activity ofsemaphorin in a collagen gel (claim 8); the nerve regeneration promoteraccording to claim 8, wherein the compound having suppressing activityfor the growth cone collapse activity and/or suppressing action on thenerve outgrowth inhibitory activity in a collagen gel exhibits saidsuppressing action at a concentration of 100 μg/ml or below (claim 9);the nerve regeneration promoter according to claim 9, wherein thecompound having suppressing activity for the growth cone collapseactivity and/or suppressing action on the nerve outgrowth inhibitoryactivity in a collagen gel exhibits said suppressing action at aconcentration of 10 μg/ml or below (claim 10); the nerve regenerationpromoter according to claim 10, wherein the compound having suppressingactivity on the growth cone collapse activity and/or suppressing actionon the nerve outgrowth inhibitory activity in a collagen gel exhibitssaid suppressing action at a concentration of 3 μg/ml or below (claim11); the nerve regeneration promoter according to any of claims 2-11,wherein the semaphorin inhibitor is a compound which inhibits thefunction of semaphorin by contacting said semaphorin (claim 12); thenerve regeneration promoter according to any of claims 2-12, wherein thesemaphorin inhibitor is a compound which does not substantially affectcell proliferation (claim 13); the nerve regeneration promoter accordingto any of claims 2-13, wherein the semaphorin inhibitor is a compoundwith a molecular weight of 1000 or less (claim 14); the nerveregeneration promoter according to any of claims 2-14, wherein thesemaphorin inhibitor is a non-peptide and a non-nucleotide compound(claim 15); the nerve regeneration promoter according to claim 15,wherein the non-peptide and the non-nucleotide compound is obtained fromcultivating Penicillium sp. SPF-3059 (claim 16); and the nerveregeneration promoter according to claim 16, wherein the non-peptide andthe non-nucleotide compound obtained from cultivating Penicillium sp.SPF-3059 contains a group represented by formulae [1], [2], [4] or [5]and/or a group represented by formulae [6] or [7] in the molecule,

[0009] represents a single bond or a double bond, R¹ represents ahydrogen atom, a carboxyl group or an alkoxycarbonyl group and R²represents a hydrogen atom, a hydroxyl group or an acyloxy group,

[0010] wherein R¹ and R² have the same meanings as in formula [1] and R³represents a hydrogen atom, a methoxymethyl group or formula [3],

[0011] wherein R¹ and R² have the same meanings as in formula [1],

[0012] wherein R⁴ represents a hydrogen atom, a carboxyl group or analkoxycarbonyl group and R⁵ represents a hydrogen atom, a hydroxyl groupor an acyloxy group,

[0013] wherein R⁴ and R⁵ have the same meanings as in formula [6] (claim17).

[0014] The present invention further relates to: a semaphorin inhibitorcontaining a compound which has a group represented by formulae [1],[2], [4] or [5] and a group represented by formulae [6] or [7] in themolecule, a pharmaceutically acceptable salt thereof or a derivativethereof, as an active ingredient,

[0015] represents a single bond or a double bond, R¹ represents ahydrogen atom, a carboxyl group or an alkoxycarbonyl group and R²represents a hydrogen atom, a hydroxyl group or an acyloxy group,

[0016] wherein R¹ and R² have the same meanings as in formula [1] and R³represents a hydrogen atom, a methoxymethyl group or formula [3],

[0017] wherein R¹ and R² have the same meanings as in formula [1],

[0018] wherein R⁴ represents a hydrogen atom, a carboxyl group or analkoxycarbonyl group and R⁵ represents a hydrogen atom, a hydroxyl groupor an acyloxy group,

[0019] wherein R⁴ and R⁵ have the same meanings as in formula [6] (claim18); a semaphorin inhibitor containing a compound represented by formula[8], a pharmaceutically acceptable salt thereof or a derivative thereof,as an active ingredient,

[0020] wherein R⁴ and R⁵ have the same meanings as in formula [6] and R⁶and R⁷ are represented by either (1) or (2) below: (1) R⁶ represents amethyl group and R⁷ represents a group shown by formulae [2], [9] or[10],

[0021] wherein R¹ and R² have the same meanings as in formula [1] and R³represents a hydrogen atom, a methoxymethyl group or formula [3],

[0022] wherein R¹ and R² have the same meanings as in formula [1],

[0023] wherein R¹ and R² have the same meanings as in formula [1], (2)R⁶ represents a group shown by formulae [5] or [11] and R⁷ represents anacetyl group,

[0024] (claim 19); a semaphorin inhibitor containing a compoundrepresented by formula [12], a pharmaceutically acceptable salt thereofor a derivative thereof, as an active ingredient,

[0025] wherein R¹, R², R⁴ and R⁵ have the same meanings as in formulae[1] and [6] (claim 20); the semaphorin inhibitor according to claim 20which contains a compound wherein at least one of R² and R⁵ represents ahydroxyl group in formula [12], a pharmaceutically acceptable saltthereof or a derivative thereof, as an active ingredient (claim 21); thesemaphorin inhibitor according to claim 21 which contains a compoundwherein R² represents a hydroxyl group in formula [12], apharmaceutically acceptable salt thereof or a derivative thereof, as anactive ingredient (claim 22); the semaphorin inhibitor according toclaim 21 which contains a compound wherein R² and R⁵ represent ahydroxyl group in formula [12], a pharmaceutically acceptable saltthereof or a derivative thereof, as an active ingredient (claim 23); thesemaphorin inhibitor according to any of claims 20-23 which contains acompound wherein R⁴ represents a carboxyl group in formula [12], apharmaceutically acceptable salt thereof or a derivative thereof, as anactive ingredient (claim 24); the semaphorin inhibitor according toclaim 20 which contains a compound wherein R¹ and R⁴ represent acarboxyl group and R² represents a hydroxyl group in formula [12], apharmaceutically acceptable salt thereof or a derivative thereof, as anactive ingredient (claim 25); and a nerve regeneration promotercontaining the semaphorin inhibitor according to any of claims 18-25 asan active ingredient (claim 26).

[0026] The present invention still further relates to: a compoundrepresented by formula [12], wherein at least one of R¹, R², R⁴ and R⁵is represented by a hydrogen atom, a pharmaceutically acceptable saltthereof or a derivative thereof,

[0027] wherein R¹, R², R⁴ and R⁵ have the same meanings as in formulae[1] and [6] (claim 27); the compound according to claim 27 wherein atleast one of R² and R⁵ represents a hydroxyl group, a pharmaceuticallyacceptable salt thereof or a derivative thereof (claim 28); the compoundaccording to claim 28 wherein R² represents a hydroxyl group, apharmaceutically acceptable salt thereof or a derivative thereof (claim29); the compound according to claim 28 wherein R² and R⁵ represent ahydroxyl group, a pharmaceutically acceptable salt thereof or aderivative thereof (claim 30); the compound according to any of claims27-30 wherein R⁴ represents a carboxyl group, a pharmaceuticallyacceptable salt thereof or a derivative thereof (claim 31); and thecompound according to claim 27 wherein R¹ and R⁴ represent a carboxylgroup and R² represents a hydroxyl group, a pharmaceutically acceptablesalt thereof or a derivative thereof (claim 32).

[0028] The present invention further relates to: a semaphorin inhibitorcontaining a compound represented by formula [13], a pharmaceuticallyacceptable salt thereof or a derivative thereof, as an activeingredient,

[0029] wherein R¹, R², R⁴ and R⁵ have the same meanings as in formulae[1] and [6] (claim 33); the semaphorin inhibitor according to claim 33which contains a compound wherein at least one of R² and R⁵ represents ahydroxyl group in formula [13], a pharmaceutically acceptable saltthereof or a derivative thereof, as an active ingredient (claim 34); thesemaphorin inhibitor according to claim 34 which contains a compoundwherein R² represents a hydroxyl group in formula [13], apharmaceutically acceptable salt thereof or a derivative thereof, as anactive ingredient (claim 35); the semaphorin inhibitor according toclaim 34 which contains a compound wherein R² and R⁵ represent ahydroxyl group in formula [13], a pharmaceutically acceptable saltthereof or a derivative thereof, as an active ingredient (claim 36); thesemaphorin inhibitor according to any of claims 33-36 which contains acompound wherein R⁴ represents a carboxyl group in formula [13], apharmaceutically acceptable salt thereof or a derivative thereof, as anactive ingredient (claim 37); the semaphorin inhibitor according toclaim 33 which contains a compound wherein R² and R⁵ represent ahydroxyl group, R¹ represents a carboxyl group and R⁴ represents ahydrogen atom in formula [13], a pharmaceutically acceptable saltthereof or a derivative thereof, as an active ingredient (claim 38); thesemaphorin inhibitor according to claim 33 which contains a compoundwherein R¹ and R⁴ represent a carboxyl group and R⁵ represents ahydroxyl group in formula [13], a pharmaceutically acceptable saltthereof or a derivative thereof, as an active ingredient (claim 39); anda nerve regeneration promoter containing the semaphorin inhibitoraccording to any of claims 33-39 as an active ingredient (claim 40).

[0030] The present invention still further relates to: a compoundrepresented by formula [13], wherein at least one of R¹, R² and R⁵represents a hydrogen, a pharmaceutically acceptable salt thereof or aderivative thereof,

[0031] wherein R¹, R², R⁴ and R⁵ have the same meanings as in formulae[1] and [6] (claim 41); the compound according to claim 41 wherein atleast one of R² and R⁵ represents a hydroxyl group, a pharmaceuticallyacceptable salt thereof or a derivative thereof (claim 42); the compoundaccording to claim 42 wherein R² represents a hydroxyl group, apharmaceutically acceptable salt thereof or a derivative thereof (claim43); the compound according to claim 42 wherein R² and R⁵ represent ahydroxyl group, a pharmaceutically acceptable salt thereof or aderivative thereof (claim 44); the compound according to any of claims41-44 wherein R⁴ represents a carboxyl group, a pharmaceuticallyacceptable salt thereof or a derivative thereof (claim 45); the compoundaccording to claim 41 wherein R² represents a hydroxyl group and R⁴represents a carboxyl group, a pharmaceutically acceptable salt thereofor a derivative thereof (claim 46); the compound according to claim 41wherein R¹ and R⁴ represent a carboxyl group and R⁵ represents ahydroxyl group, a pharmaceutically acceptable salt thereof or aderivative thereof (claim 47); a compound represented by formula [14], apharmaceutically acceptable salt thereof or a derivative thereof,

[0032] wherein R¹, R², R³, R⁴ and R⁵ have the same meanings as informulae [2] and [6] (claim 48); the compound according to claim 48wherein at least one of R² and R⁵ represents a hydroxyl group, apharmaceutically acceptable salt thereof or a derivative thereof (claim49); the compound according to claim 49 wherein R² represents a hydroxylgroup, a pharmaceutically acceptable salt thereof or a derivativethereof (claim 50); the compound according to claim 49 wherein R² and R⁵represent a hydroxyl group, a pharmaceutically acceptable salt thereofor a derivative thereof (claim 51); the compound according to any ofclaims 48-51 wherein R⁴ represents a carboxyl group, a pharmaceuticallyacceptable salt thereof or a derivative thereof (claim 52); the compoundaccording to claim 48 wherein R¹ and R⁴ represent a carboxyl group andR² and R⁵ represent a hydroxyl group, a pharmaceutically acceptable saltthereof or a derivative thereof (claim 53); the compound according toclaim 48 wherein R¹ represents a carboxyl group, R² and R⁵ represent ahydroxyl group and R³ represents a methoxymethyl group, apharmaceutically acceptable salt thereof or a derivative thereof (claim54); the compound according to claim 48 wherein R¹ represents a carboxylgroup or a methoxycarbonyl group, R⁴ represents a carboxyl group, R³represents a hydrogen atom and R⁵ represents a hydroxyl group, apharmaceutically acceptable salt thereof or a derivative thereof (claim55); the compound according to claim 55 wherein R¹ represents a carboxylgroup or a methoxycarbonyl group, R⁴ represents a carboxyl group, R² andR³ represent a hydrogen atom and R⁵ represents a hydroxyl group, apharmaceutically acceptable salt thereof or a derivative thereof (claim56); the compound according to any of claims 48-53, wherein R³represents a group shown by formula [3], a pharmaceutically acceptablesalt thereof or a derivative thereof

[0033] (claim 57); a semaphorin inhibitor containing a compound of anyof claims 48-57, a pharmaceutically acceptable salt thereof or aderivative thereof, as an active ingredient (claim 58); and a nerveregeneration promoter containing the semaphorin inhibitor according toclaim 58 as an active ingredient (claim 59).

[0034] The present invention still further relates to: a compoundrepresented by formula [15], a pharmaceutically acceptable salt thereofor a derivative thereof,

[0035] wherein R⁴ and R⁵ have the same meanings as in formula [6] (claim60); the compound according to claim 60 wherein R⁵ represents a hydroxylgroup, a pharmaceutically acceptable salt thereof or a derivativethereof (claim 61); the compound according to claim 60 or 61 wherein R⁴represents a carboxyl group, a pharmaceutically acceptable salt thereofor a derivative thereof (claim 62); a semaphorin inhibitor containing acompound of any of claims 60-62, a pharmaceutically acceptable saltthereof or a derivative thereof, as an active ingredient (claim 63); anda nerve regeneration promoter containing the semaphorin inhibitor ofclaim 63 as an active ingredient (claim 64).

[0036] The present invention still further relates to: a compoundrepresented by formula [16], a pharmaceutically acceptable salt thereofor a derivative thereof,

[0037] wherein R¹, R², R⁴ and R⁵ have the same meanings as in formulae[1] and [6] (claim 65); the compound according to claim 65 wherein atleast one of R² and R⁵ represents a hydrogen atom, a pharmaceuticallyacceptable salt thereof or a derivative thereof (claim 66); the compoundaccording to claim 65 wherein R² and R⁵ represent a hydroxyl group, apharmaceutically acceptable salt thereof or a derivative thereof (claim67); the compound according to any of claims 65-67 wherein R⁴ representsa carboxyl group, a pharmaceutically acceptable salt thereof or aderivative thereof (claim 68); a semaphorin inhibitor containing acompound of any of claims 65-68, a pharmaceutically acceptable saltthereof or a derivative thereof, as an active ingredient (claim 69); anda nerve regeneration promoter containing the semaphorin inhibitor ofclaim 69 as an active ingredient (claim 70).

[0038] The present invention still further relates to: a preventive or aremedy for neuropathic diseases and/or neurodegenerative diseasescontaining the nerve regeneration promoter of any of claims 1-17, 26,40, 59, 64 or 70 (claim 71); the preventive or the remedy forneuropathic diseases and/or neurodegenerative diseases according toclaim 71, wherein said neuropathic diseases and/or neurodegenerativediseases are accompanied with a spinal nerve injury and/or a peripheralnerve injury (claim 72); the preventive or the remedy for neuropathicdiseases and/or neurodegenerative diseases according to claim 71,wherein said neuropathic diseases and/or neurodegenerative diseases areolfactory abnormality, traumatic neuropathy, cerebral infarctionalneuropathy, facial nerve paralysis, diabetic neuropathy, glaucoma,retinitis pigmentosa, Alzheimer's disease, Parkinson's disease,neurodegenerative diseases, muscular hypoplastic lateral sclerosis, LouGehrig's disease, Huntington's chorea, cerebral infarction or traumaticneurodegenerative diseases (claim 73); a process for producing thecompound according to any of claims 27-32, wherein the process comprisescultivating a compound-producing fungus of any of claims 27-32 whichbelongs to the genus Penicillium and collecting said compound from theculture (claim 74); the process for producing the compound according toany of claims 41-47, wherein the process comprises cultivating acompound-producing fungus of any of claims 41-47 which belongs to thegenus Penicillium and collecting said compound from the culture (claim75); the process for producing the compound according to any of claims48-57, wherein the process comprises cultivating a compound-producingfungus of any of claims 48-57 which belongs to the genus Penicillium andcollecting said compound from the culture (claim 76); the process forproducing the compound according to any of claims 60-62, wherein theprocess comprises cultivating a compound-producing fungus of any ofclaims 60-62 which belongs to the genus Penicillium and collecting saidcompound from the culture (claim 77); the process for producing thecompound according to any of claims 65-68, wherein the process comprisescultivating a compound-producing fungus of any of claims 65-68 whichbelongs to the genus Penicillium and collecting said compound from theculture (claim 78); the production process according to any of claims74-78, wherein the producing fungus which belongs to the genusPenicillium is Penicillium sp. SPF-3059 (claim 79); and Penicillium sp.SPF-3059 (FERM BP-7663) or a fungus strain induced from said SPF-3059(claim 80).

BRIEF DESCRIPTION OF DRAWINGS

[0039]FIG. 1 shows the concentration-dependent inhibitory action of thesemaphorin inhibitors of the present invention, SPF-3059-1, M162 andA721, on the growth cone collapse activity of Sema3A.

[0040]FIG. 2 shows the concentration-dependent inhibitory action of thesemaphorin inhibitors of the present invention, SPF-3059-1, SPF-3059-2and SPF-3059-5, on the growth cone collapse activity of Sema3A.

[0041]FIG. 3 shows the concentration-dependent inhibitory action of thesemaphorin inhibitor SPF-3059-1 of the present invention on the growthcone collapse activity of Sema6C.

[0042]FIG. 4 is photographs showing inhibitory action of the semaphorininhibitors of the present invention, SPF-3059-1, M162 and A721, on theneurite outgrowth inhibitory activity of Sema3A in the collagen-gelco-culture method.

[0043]FIG. 5 shows the in vivo nerve-regeneration promoting action ofthe semaphorin inhibitor SPF-3059-1 of the present invention in theolfactory nerve incision.

[0044]FIG. 6 shows the in vivo nerve-regeneration promoting action ofthe semaphorin inhibitors M162 and A721 of the present invention in theolfactory nerve incision.

[0045]FIG. 7 shows the nerve-regeneration promoting action of thesemaphorin inhibitor SPF-3059-1 of the present invention in the sciaticnerve wound.

[0046]FIG. 8 shows the result of examining influence of the semaphorininhibitor SPF-3059-1 of the present invention on the proliferation ofCOS7 cells.

[0047]FIG. 9 shows the result of examining influence of the semaphorininhibitor SPF-3059-1 of the present invention on the semaphorin 3Areceptor (Neuropilin-1) binding.

[0048]FIG. 10 shows the result of examining that the target molecule ofthe semaphorin inhibitor SPF-3059-1 of the present invention issemaphorin 3A.

[0049]FIG. 11 is photographs showing the results of examining the invivo nerve-regeneration promoting action of the semaphorin inhibitorSPF-3059-1 of the present invention in the spinal nerve (cerebralcortex-spinal cord pathway) incision.

[0050]FIG. 12 is photographs showing the results of examining the invivo nerve-regeneration promoting action of PBS as a control in thespinal nerve (cerebral cortex-spinal cord pathway) incision.

BEST MODE OF CARRYING OUT THE INVENTION

[0051] There is no particular limitation to a nerve regenerationpromoter of the present invention as long as the promoter contains aninhibitor for a nerve outgrowth-repelling factor such as semaphorin orthe like as an active ingredient. Here, semaphorin is a generic name forproteins that have semaphorin domains with similar structures consistingof about 500 amino acid residues (Neuron 14, 941-948, 1995), andapproximately 20 variants or more have been reported to date. In thepresent invention, however, semaphorins will not be limited to thesepublicly known. The following can be exemplified as such semaphorins:semaphorins of mammals such as human, etc.; preferably class 3, 4, 5 or6 semaphorins as defined in the literature (Cell 97, 551, 1999); morepreferably class 3 or 6 semaphorins; and most preferably semaphorin 3A(Cell 75, 217, 1993, Cell 75,1389, 1993) in class 3 semaphorins andsemaphorin 6C (WO98/11216, Moll. Cell. Neurosci. 13, 9-23 (1999)) inclass 6 semaphorins. As sequence information regarding genes encodingthese semaphorins is disclosed in GenBank data base, the aforementionedliteratures or the like, cDNA encoding semaphorin can be clonedaccording to the said sequence information and by using, for example, abrain-derived cDNA library or the like with an appropriate portion ofDNA as a probe for hybridization or as a primer for PCR. Those skilledin the art can easily perform these cloning according to basic protocolssuch as Molecular Cloning 2nd Ed., Cold Spring Harbor Laboratory Press(1989), etc. Besides, proteins can be produced according to varioustextbooks and literatures such as the aforementioned Molecular Cloningor the like by expressing a gene encoding the semaphorin thus obtained.Further, semaphorin of the present invention will not be limited only toa natural or a recombinant protein but also includes: a protein in whichthe extracellular domain of a membrane-bound semaphorin is expressed andsolubilized; a fusion protein with other protein such as an antibody,alkaline phosphatase or the like; a protein to which a tag such asHis-tag, Flag or the like is added; or a mutant in which part of aminoacids are deleted, substituted or added.

[0052] For example, semaphorin 6C (Sema6C) is a membrane-bound proteinand the extracellular domain of Sema6C is usually used such as formeasuring the activity of a subject substance by utilizing promoting orsuppressing actions of Sema6C activities. Two isoforms are known in theextracellular domain of Sema6C (WO98/11216 and Moll. Cell. Neurosci. 13,9-23 (1999)), each of which has the growth cone collapse activity. Afusion protein in which the extracellular domain of said Sema6C and amarker protein and/or a peptide tag are bound can be advantageously usedfor such cases of measuring the activity of a subject substance as longas the activity of Sema6C is not impaired. Examples of marker proteinsare conventionally well known marker proteins such as alkalinephosphatase (Cell 63, 185-194 (1990)), Fc region of an antibody (Genbankaccession number M87789), HRP and the like, and peptide tags areexemplified by conventionally well known Mic-tag, His-tag, Flag-tag andthe like.

[0053] In the present invention, a semaphorin inhibitor means asubstance which inhibits an activity of any one of the aforementionedsemaphorins such as, for instance, migration activity of a cell, celldeath-inducing activity, morphological changes of a cell such as cellrounding or growth cone collapsing, suppressing or promoting activityfor neurite outgrowth, suppressing or promoting activity for dendriteoutgrowth of nerve cells, nerve axon guidance activity or the like. Anysubstance inhibiting the foregoing semaphorin activities can be adoptedas said semaphorin inhibitor without particular limitation. Preferably,a compound having promoting action on central and/or peripheral nerveregeneration, more preferably a compound having suppressing action onthe growth cone collapse activity and/or the nerve outgrowth inhibitoryactivity in a collagen gel, and even more preferably a compound havingsuppressing action on both growth cone collapse activity of semaphorinand nerve outgrowth inhibitory activity in a collagen gel areexemplified.

[0054] The above described promoting action on the central and/orperipheral nerve regeneration refers to an action which promotes nerveregeneration in the central nervous system (tissue) comprising such asbrain, spinal cord and the like, and/or in the peripheral nervous system(tissue) which are in organs on body surface or in the body that consistthe marginal and peripheral portions and not the said central nervoussystem (tissue). Here, promoting action on the central nerveregeneration includes promoting action not only on the nerveregeneration wherein an axon emerges from a nerve cell body in thecentral region, such as a retinal nerve or a cerebral cortex nerve, andis projected on other nerve cell which is also in the central, but alsoon the nerve regeneration wherein a nerve axon is regenerated in thecentral nervous system (tissue) circumstance even when a nerve, such asan afferent fiber of an olfactory nerve or a dorsal root ganglionsensory nerve, emerges from a nerve cell body in the peripheral.Further, promoting action on the peripheral nerve regeneration includespromoting action not only on the nerve regeneration of a nerve whichemerges from a peripheral nerve cell body and extends in a peripheraltissue, but also on the nerve regeneration wherein the circumstance forregeneration is the peripheral nervous system (tissue) even when a nerveis emerged from a central nerve cell body (brain, spinal cord or thelike). The latter can be exemplified by the nerve-regeneration promotingaction for such as a spinal cord motor nerve, a preganglionic nerve inthe autonomic nervous systems like sympathetic and parasympatheticnerves, and the like. Promoting action on regeneration of a nerve suchas a sciatic nerve, which has both nerves mentioned above, is alsoincluded in the exemplification. A compound with promoting action on thecentral and peripheral nerve regeneration is particularly preferable fora semaphorin inhibitor of the present invention. The central nervoussystem (tissue) described earlier refers to a tissue comprising brain,medulla oblongata, spinal cord, eye and the like, and more particularlyrefers to a region where the transport of polymer substances isrestricted by structures such as the blood-brain barrier and theblood-retina barrier. The peripheral nervous system (tissue) refers tothe region of the other parts of the body. Nerve fibers are in generalcapable of regenerating in the peripheral nervous tissues, but they areunable to regenerate in the central nervous tissues.

[0055] The growth cone collapse activity of semaphorin described abovemeans an activity to make growth cones disappear. This activity isobserved after performing the following steps: cultivating nerve cells(generally tissue explants of ganglions) for a given period of time invitro until the extended neurites as well as the growth cones at theedge of said neurites can be observed; and then adding thereto a givenconcentration (e.g. about 3 unit/ml; 1 unit/ml is defined as asemaphorin concentration in which 50% of the growth cones are collapsed)of semaphorin and cultivating for another given period of time (e.g. onehour). In order to get the extended neurites and the growth cones at theedge of said neurites ready for the observation, the nerve cells aregenerally cultivated for 10 to 20 hours in vitro, which duration can bealtered according to a nerve variant and culture conditions. When thegrowth cone collapse caused by semaphorin is suppressed by, for example,the addition of a compound to this experimental system at an appropriateconcentration about one hour prior to the addition of semaphorin, thensuch compound is regarded as a semaphorin inhibitor, especially as acompound with suppressing action on the growth cone collapse activity ofsemaphorin. Although there is no particular limitation to a compoundwith such suppressing action on the growth cone collapse activity,compounds can be exemplified which exhibit the said suppressing actionat a concentration of 100 μg/ml or below, preferably 30 μg/ml or below,more preferably 10 μg/ml or below, and most preferably 3 μg/ml or below.Further, a compound which does not substantially affect proliferation ofthe cells such as nerve cells, semaphorin-expressing cells or the likeis preferable as a semaphorin inhibitor in order to confirm the effectof semaphorin inhibitors of the present invention and in view of safetywhen used as a pharmaceutical.

[0056] The nerve outgrowth inhibitory activity of semaphorin in acollagen gel as described above means, for instance, the neuriteoutgrowth inhibitory activity observed in a collagen gel which contains,for example, both semaphorin-producing cells and nerve cells (usuallyganglions). And suppressing action of said neurite outgrowth inhibitoryactivity is an activity to persistently inhibit the semaphorin activityin a collagen gel, and, for instance, is an activity by which neuritescan outgrow to the side of semaphorin-producing cells as much as ½ ormore of the outgrowth observed at the opposite side in the presence ofthe object substance under the experimental condition where neurites canonly outgrow up to ⅓ or less to semaphorin producing cells compared withthe growth observed at the opposite side of the semaphorin-producingcells when observed after cultivating semaphorin-producing cells andnerve cells adjacently in a collagen gel, usually for overnight or evenlonger. Further, there is no particular limitation to a compound withsuppressing action on the neurite outgrowth inhibitory activity ofsemaphorin in said collagen gel. However, those exhibiting the foregoingsuppressing action at a concentration of 100 μg/ml or less, preferably30 μg/ml or less, more preferably 10 μg/ml or less and most preferably 3μg/ml or less are exemplified.

[0057] Semaphorin used for measuring the two types of semaphorinactivities mentioned above is not limited to a natural semaphorin andthe following semaphorins described earlier can be used as well:semaphorin in which only the extracellular domain of a membrane-bindingsemaphorin is expressed and solubilized; a fusion protein with otherprotein such as an antibody, alkaline phosphotase or the like;semaphorin to which a tag such as a His-tag or a Flag is added; orsemaphorin in which some amino acids are altered. Further, dorsal rootganglions from chick embryos of 7 or 8 embryonic days are convenientlyused as nerve cells for the culture. However, dorsal root ganglions ofanimals other than chicks, or any other nerve cells such as sympatheticganglions, retinal ganglions, superior cervical ganglions or the likeother than dorsal root ganglions may also be used as long as the nervecells are capable of extending their neurites in the in vitro culture.There is no particular limitation to the culture condition as long asneurite outgrowth can be observed and semaphorin activities can bemeasured.

[0058] The action mechanism of the semaphorin inhibitors of the presentinvention can be considered as follows. The neurite outgrowth inhibitionor the growth cone collapse caused by semaphorin is triggered by bindingof semaphorin to its receptor on the nerve cell surface (growth cone).The signal is transmitted from the receptor to which semaphorin is boundto the intracellular signaling pathway and depolymerization of actinfibers is finally raised, which as a result gives rise to the neuriteoutgrowth suppression and the growth cone collapse. Inhibition ofsemaphorin activity is achieved by inhibiting or blocking any of thesteps in the course of these reactions. As the above-mentioned receptorfor semaphorin, a receptor for any of the foregoing semaphorins may beadopted, and a mutant or a component of a part of such receptor may alsobe adopted provided that semaphorin can bind to it. The examples areNeuropilin-1, plexin and the like. The semaphorin inhibitors of thepresent invention will not be restricted by their action mechanisms andan inhibitor which inhibits any one of the steps in the above-describedaction mechanism is included in the category of the present invention.That is to say, a compound is also included in the category of thepresent invention, when the compound inhibits semaphorin activity byinhibiting the reactions concerning the intracellular signaling pathwaywhich takes place from the aforementioned receptor-binding of semaphorinto the depolymerization of actin fibers. Besides, a method of measuringthe receptor-binding inhibitory activity of semaphorin can be any methodif appropriately selected by those skilled in the art, which isexemplified by a method of measuring the receptor-binding inhibitoryactivity of semaphorin wherein semaphorin fused with other protein suchas an antibody, alkaline phosphatase or the like or semaphorin to whichHis-tag, Flag or the like is added, as described earlier, is bound to areceptor of said semaphorin or to a cell which expresses a receptorcomponent in the presence of a subject substance.

[0059] For example, the inhibitory activity of SPF-3059-1 for thebinding of Sema3A to Neuropilin-1 was actually examined by using thealkaline phosphatase-fused Sema3A (=Sema3A-AP) andNeuropilin-1-expressing COS7 cells. SPF-3059-1 is a compound discoveredby the present inventors, which inhibits both collapse and neuriteoutgrowth inhibition induced by semaphorin 3A (Sema3A). The presentstudy revealed that SPF-3059-1 inhibits the binding of Sema3A-AP toNeuropilin-1 in a concentration-dependent manner. Further, in order toelucidate the mechanism of the said inhibition, that is, in order toelucidate whether SPF-3059-1 interacts with Sema3A or with its receptor,the comparison was made by measuring collapse activity between (1) thecollapse activity which was observed when a sample, to which SPF-3059-1and Sema3A were mixed in advance at the concentration raising sufficientinhibitory activity (0.25 μg/ml of SPF-3059-1), was added to the culturesolution of dorsal root ganglions and (2) the collapse activity whichwas observed when Sema3A was added to the above-mentioned culturesolution after the addition of SPF-3059-1. The final concentration ofSPF-3059-1 (0.05 μg/ml) in the culture solution was the same both for(1) and (2). Inhibition of collapse activity was not observed in (2) atthis concentration of SPF-3059-1. However in (1), inhibition of collapseactivity was observed. This result indicates that Sema3A has lost itscollapse activity because SPF-3059-1 interacted with it. From the aboveconsiderations, SPF-3059-1 is thought to act by a mechanism in which thebinding of Sema3A to the receptor is inhibited by the direct action ofSPF-3059-1 on Sema3A. When this finding is considered, a compound whichinhibits the binding of semaphorin to its receptor by interactingdirectly with the semaphorin and which inhibits the function and theactivity of the semaphorin is preferable for a semaphorin inhibitor ofthe present invention. A semaphorin inhibitor, particularly a compoundwhich inhibits the function of semaphorin by interacting with it, can bescreened by the above mentioned methods of measuring the collapseactivity or the receptor-binding inhibitory activity, or the like.

[0060] A preferable compound for the semaphorin inhibitor of the presentinvention is a compound where the semaphorin inhibitor will notsubstantially affect cell proliferation, i.e. a compound not showing thesuppressing action on cell proliferation, for example, a compound whichdoes not show the suppressing action on cell proliferation at aconcentration which is 50-3000 folds or higher than the concentration atwhich the semaphorin inhibitory activity can be observed. Further,although there is no particular limitation to molecular weight ofsemaphorin inhibitors of the present invention, a compound with a lowmolecular weight is desirable in view of diffusibility, membranepermeability, tissue distribution, especially blood-brain barrierpermeability or the like, and suitable examples include a low molecularweight compound with a molecular weight of 10000 or less, preferably amolecular weight of 5000 or less, more preferably a molecular weight of1000 or less and particularly a compound with a molecular weight of 600or less. Still further, as for semaphorin inhibitors of the presentinvention, non-peptide and non-nucleotide compounds can be exemplified.Said non-peptide and non-nucleotide compounds are exemplified by analiphatic synthesized compound M162, an actinomycete-derived compoundA721, and most preferably, compounds obtained from the culture ofPenicillium sp. SPF-3059 which is described later.

[0061] An example of the above-mentioned compounds obtained from theculture of Penicillium sp. SPF-3059 is a compound with semaphorininhibitory activity which contains in its chemical structure a grouprepresented by the above-mentioned general formulae [1], [2], [4] or [5]and/or a group represented by the above-mentioned general formula [6] or[7], preferably a compound represented by the above-mentioned generalformula [8] and more preferably a compound represented by theabove-mentioned general formula either [12] to [16]. In the generalformulae [1], [2], [4], [9], [10], [11], [12], [13], [14] and [16] inthese compounds, R¹ represents a hydrogen atom, a carboxyl group or analkoxycarbonyl group, preferably a hydrogen atom or a carboxyl group,and a methoxycarbonyl group, an ethoxycarbonyl group, a propoxycarbonylgroup and the like are exemplified as the above-mentioned alkoxycarbonylgroup, among which a methoxycarbonyl group is preferable. Particularly,R¹ in the general formulae [1], [4], [9], [10], [11], [12], [13] and[16] preferably represent a hydrogen atom or a carboxyl group and R¹ inthe general formulae [2] and [14] preferably represent a hydrogen atom,a carboxyl group or a methoxycarbonyl group where a hydrogen atom or acarboxyl group is more preferable. Similarly, R² in the general formulae[1], [2], [4], [9], [10], [11], [12], [13], [14] and [16] represent ahydrogen atom, a hydroxyl group or an acyloxy group, preferably ahydrogen atom or a hydroxyl group. As the above-mentioned acyloxy group,an acetoxy group, a propionyloxy group, a pivaloyloxy group and the likeis exemplified as the above-mentioned acyloxy group. R³ in the generalformulae [2] and [14] represent a hydrogen atom, a methoxymethyl groupor a group shown by the formula [3]. R⁴ in the general formulae [6],[7], [8], [12], [13], [14], [15] and [16] represent a hydrogen atom, acarboxyl group or an alkoxycarbonyl group, preferably a hydrogen atom ora carboxyl group. The examples of the above-mentioned alkoxycarbonylgroup are a methoxycarbonyl group, an ethoxycarbonyl group, apropoxycarbonyl group and the like, among which a methoxycarbonyl groupis preferable. Similarly, R⁵ in the general formulae [6], [7], [8],[12], [13], [14], [15] and [16] represent a hydrogen atom, a hydroxylgroup or an acyloxy group, preferably a hydrogen atom or a hydroxylgroup. And an acetoxy group, a propionyloxy group, a pivaloyloxy groupand the like is exemplified as the above-mentioned acyloxy group. As toR⁶ and R⁷ in the general formula [8], (1) when R⁶ represents a methylgroup, R⁷ represents a group shown by the formulae [2], [9] or [10] and(2) when R⁶ represents a group shown by the formula [5] or [11], R⁷represents an acetyl group.

[0062] A compound having a group represented by the above-mentionedgeneral formulae [1], [2], [4] or [5] and/or a group represented by thegeneral formula [6] or [7] in the molecule as described above,preferably a compound represented by the above-mentioned general formula[8] and particularly a compound represented by the above-mentionedgeneral formulae [12] to [16] can be obtained from the culture ofPenicillium sp. SPF-3059 with the semaphorin inhibitory activity as anindex. Moreover, such compound can also be obtained from the compoundwith semaphorin inhibitory activity thus obtained or the like by knownconverting and synthetic methods with the semaphorin inhibitory-activityas an index. Compounds which constitute semaphorin inhibitors of thepresent invention are exemplified by those shown by the formulae[17]-[37] which are described later in the Examples in this description,and are more specifically exemplified by: the compounds represented bythe general formula [12] which is shown by the formulae [17], [20],[23], [24] and [32]; the compounds represented by the general formula[13] shown by the formulae [18], [19], [21], [25], [26], [27] and [28];the compounds represented by the general formula [14] shown by theformulae [22], [30], [31], [34], [36] and [37]; the compoundsrepresented by the general formula [15] shown by the formulae [29] and[35]; and the compounds represented by the general formula [16] shown bythe formula [33]. These compounds specifically exemplified in the aboveare novel compounds except for the following: the compound SPF-3059-1shown by the formula [17] (Japanese Laid-Open Patent ApplicationNo.1993-239050); the compound SPF-3059-2 shown by the formula [18] (Pure& Appl. Chem. 66, 2383-2386, 1994); and the compound SPF-3059-5 shown bythe formula [19] (Pure & Appl. Chem. 66, 2383-2386, 1994, and PublishedJapanese translation of PCT international publication No.1994-506202).

[0063] Further, in the compounds which constitute semaphorin inhibitorsof the present invention, salts or derivatives of the compounds,preferably pharmaceutically or veterinary pharmaceutically acceptablesalts or derivatives will also be included in the category of thepresent invention. The examples of salts are: inorganic basic salts suchas sodium salt, potassium salt, calcium salt, magnesium salt, aluminumsalt, ammonium salt or the like; organic basic salts such astriethylammonium salt, triethanolammonium salt, pyridinium salt,diisopropylammonium salt or the like; basic amino acid salts such asarginine salt, lysine salt or the like. The derivatives are exemplifiedthose in which the carboxyl group or the hydroxyl group of a compound isconverted to an ester group where the examples include a derivative inwhich the hydroxyl group is acylated by an acyl group with 2 to 5carbons such as an acetyl group, a propionyl group or the like, and aderivative in which the carboxyl group is converted to esters with 2 to5 carbons such as methylester, ethylester, or the like.

[0064] Examples of a compound of the present invention, preferably acompound having semaphorin inhibitory activity and particularly acompound obtained from the culture of Penicillium sp. SPF-3059 andhaving semaphorin inhibitory activity include a compound wherein atleast one of R¹, R², R⁴ and R⁵ in the above-mentioned general formula[12] (wherein R¹, R², R⁴ and R⁵ are as described earlier) is representedby a hydrogen atom, preferably a compound wherein at least one of R² andR⁵ represents a hydroxyl group, for instance, a compound in which R²represents a hydroxyl group (a compound in which R² and R⁵ represent ahydroxyl group and a hydrogen atom, respectively, etc.), a compound inwhich R² and R⁵ represent a hydroxyl group, the above-mentioned compoundin which R⁴ represents a carboxyl group and the above-mentioned compoundin which R¹ and R⁴ represent a carboxyl group and R² represents ahydroxyl group and the like. It is specifically exemplified by thecompound SPF-3059-3 shown by the formula [20], the compound SPF-3059-7shown by [23], the compound SPF-3059-9 shown by [24] and the compoundSPF-3059-30 shown by [32]. Compounds of the present invention alsoinclude pharmaceutically acceptable salts or derivatives of the compoundrepresented by the above-mentioned general formula [12].

[0065] Examples of a compound of the present invention, preferably acompound having semaphorin inhibitory activity, and particularly acompound obtained from the culture of Penicillium sp. SPF-3059 andhaving semaphorin inhibitory activity include a compound wherein atleast one of R¹, R² and R in the above-mentioned general formula [13](wherein R¹, R², R⁴ and R⁵ are as described earlier) is represented by ahydrogen atom, preferably a compound wherein at least one of R² and R⁵represents a hydroxyl group, for instance, a compound in which R²represents a hydroxyl group (a compound in which R² and R⁵ represent ahydroxyl group and a hydrogen atom, respectively, etc.), a compound inwhich R² and R⁵ represent a hydroxyl group, the above-mentioned compoundin which R⁴ represents a carboxyl group and the above-mentioned compoundin which R¹ and R⁴ represent a carboxyl group and R⁵ represents ahydroxyl group and the like. It is more specifically exemplified by thecompound SPF-3059-4 shown by the formula [21], the compound SPF-3059-12shown by [25], the compound SPF-3059-24 shown by [26], the compoundSPF-3059-25 shown by [27] and the compound SPF-3059-26 shown by [28].Compounds of the present invention also include pharmaceuticallyacceptable salts or derivatives of the compound represented by theabove-mentioned general formula [13].

[0066] Examples of a compound of the present invention, preferably acompound having semaphorin inhibitory activity and particularly acompound obtained from the culture of Penicillium sp. SPF-3059 andhaving semaphorin inhibitory activity include a compound represented bythe above-mentioned general formula [14] (wherein R¹, R², R³, R⁴ and R⁵are as described earlier), preferably a compound in which at least oneof R² and R⁵ is represented by a hydroxyl group, for instance, acompound in which R² represents a hydroxyl group (a compound in which R²and R⁵ represent a hydroxyl group and a hydrogen atom, respectively,etc.), a compound in which R² and R⁵ represent a hydroxyl group, theabove-mentioned compound in which R⁴ represents a carboxyl group, acompound in which R¹ and R⁴ represent a carboxyl group and R² and R⁵represent a hydroxyl group, a compound in which R¹ represents a carboxylgroup, R² and R⁵ represent a hydroxyl group and R³ representsmethoxymethyl group, a compound in which R¹ represents either a carboxylgroup or a methoxycarbonyl group, R⁴ represents a carboxyl group, R³represents a hydrogen atom and R⁵ represents a hydroxyl group,especially a compound in which R¹ represents either a carboxyl group ora methoxycarbonyl group, R⁴ represents a carboxyl group, R² and R³represent a hydrogen atom and R⁵ represents a hydroxyl group, a compoundin which R³ represents a group shown by the above-mentioned formula [3],and so on. It is more specifically exemplified by the compoundSPF-3059-6 shown by the formula [22], the compound SPF-3059-28 shown by[30], the compound SPF-3059-29 shown by [31], the compound SPF-3059-35shown by [34], the compound SPF-3059-37 shown by [36], and the compoundSPF-3059-39 shown by [37]. Compounds of the present invention alsoinclude pharmaceutically acceptable salts or derivatives of the compoundrepresented by the above-mentioned general formula [14].

[0067] Examples of a compound of the present invention, preferably acompound having semaphorin inhibitory activity, and particularly acompound obtained from the culture of Penicillium sp. SPF-3059 andhaving semaphorin inhibitory activity include a compound represented bythe above-mentioned general formula [15] (wherein R⁴ and R⁵ are the sameas described earlier), preferably a compound in which R⁵ represents ahydroxyl group, the above-mentioned compound in which R⁴ represents acarboxyl group and the like. It is more specifically exemplified by thecompound SPF-3059-27 shown by the formula [29] and the compoundSPF-3059-36 shown by [35]. Compounds of the present invention alsoinclude pharmaceutically acceptable salts or derivatives of the compoundrepresented by the above-mentioned general formula [15].

[0068] Examples of a compound of the present invention, preferably acompound having semaphorin inhibitory activity, and particularly acompound obtained from the culture of Penicillium sp. SPF-3059 andhaving semaphorin inhibitory activity include a compound represented bythe above-mentioned general formula [16] (wherein R¹, R², R⁴ and R⁵ arethe same as described earlier), preferably a compound wherein at leastone of R² and R⁵ represents a hydroxyl group, especially a compound inwhich R² and R⁵ represent a hydroxyl group, the above-mentioned compoundin which R⁴ represents a carboxyl group and the like. It is morespecifically exemplified by the compound SPF-3059-34 shown by theformula [33]. Compounds of the present invention also includepharmaceutically acceptable salts or derivatives of the compoundrepresented by the above-mentioned general formula [16].

[0069] A compound having a group represented by the above-mentionedgeneral formulae [1], [2], [4] or [5] and/or a group represented by theabove-mentioned general formula [6] or [7] in the molecule and havingsemaphorin inhibitory activity, preferably a compound represented by theabove-mentioned general formula [8], more preferably a compoundrepresented by the above-mentioned general formula [12] to [16], morespecifically compounds constituting the semaphorin inhibitors of thepresent invention shown by the above-mentioned formulae [17] to [37],etc., all these compounds can be effectively obtained by cultivating thefungal strain SPF-3059 which belongs to the genus Penicillium. Thestrain was isolated by the present inventors from a soil samplecollected in Osaka Prefecture, Japan. The strain SPF-3059 possesses thefollowing taxonomical characteristics.

[0070] (a) Cultural and Morphological Characteristics

[0071] On malt extract agar, colonies grew slowly attaining a diameterof 2.8 to 2.9 cm in 21 days at 25.degree. C. The colonies were white toyellow and floccose in appearance. The reverse side color was darkyellow. Neither soluble pigment production nor spore formation wasobserved. On potato dextrose agar, colonies grew slowly attaining adiameter of 3.2 to 3.3 cm in 21 days at 25.degree. C. The colonies werewhite to cream yellow and floccose in appearance. The reverse side colorwas dark yellow to brown. Neither soluble pigment production nor sporeformation was observed. On Czapek agar, colonies grew slowly attaining adiameter of 3.1 to 3.2 cm in 21 days at 25° C. The colonies were whiteto gray and floccose in appearance. The reverse side color was creamyellow. Neither soluble pigment production nor spore formation wasobserved. On oatmeal agar (Actino Medium No. 3 “DAIGO”, NihonPharmaceutical Co., Ltd.), colonies grew slowly attaining a diameter of2.0 to 2.1 cm in 21 days at 25.degree. C. The colonies were white toyellow or grayish green and floccose in appearance. The reverse sidecolor was cream yellow to gray. Soluble pigment was not produced butspore formation was observed. The conidiophores were smooth-walled witha length of 5 to 20 μm, and generated 3 to 6 phialides in amonoverticillate manner at the end of the stipes. On the top of thephialides, which had a length of 3 to 4 μm, conidia were formed in achain form, with 2 to 10 conidia per chain. The conidia are globose witha diameter of 2.2 to 2.4 μm with striated surface (in general, 10longitudinal lines on the surface). Teleomorph was not observed.

[0072] (b) Physiological Characteristics

[0073] (1) pH Range for Growth

[0074] Growth was examined in shaking culture using Sabouraud broth.Observation was made after cultivation for 3 days at 27.degree. C. Theresult was as follows: pH Growth 3.1 − 4.5 + 5.5 ++ 7.1 +++ 8.0 ++ 9.0 +10.0 −

[0075] (2) Temperature Range for Growth

[0076] Growth was examined using oatmeal agar. Observation was madeafter incubation for 5 days at 38.degree. C. The result was that thestrain grew at the temperature.

[0077] Based on the above taxonomical characteristics, the strain wasidentified as a strain of the genus Penicillium and was namedPenicillium sp. SPF-3059. The said strain was deposited on Jul. 13, 2001to the International Patent Organism Depositary, the National Instituteof Advanced Industrial Science and Technology, Japan (1-1 Higashi1-chome, Tsukuba, Ibaraki 305-8566, Japan) under the accession numberFERM BP-7663 as the International Deposition Number under the BudapestTreaty on the international recognition of the deposit of microorganismsfor the purpose of patent procedure.

[0078] According to the present invention, the cultivation for theproduction of the said semaphorin inhibitors by the strain SPF-3059 canbe carried out in a nutrient medium. The nutrient medium may either beliquid or solid. Shaking culture or submerged culture with aeration ispreferable. The composition of the medium may be varied over a widerange. No particular limitation is assigned to medium composition foruse. Essentially, what is required is a carbon source, and a nitrogensource. Trace inorganic elements can also be added. Examples of suitablecarbon sources are glucose, sucrose, glycerin, starch, dextrin, molassesor the like. Examples of suitable nitrogen sources are peptone, caseinor its hydrolyzate, meat extract, yeast extract, soy bean flour, cottonseed flour, corn steep liquor, amino acids such as histidine, ammoniumsalts, nitrate salts or the like. Examples of sources of suitableinorganic elements are phosphate salts such as sodium phosphate andpotassium phosphate, magnesium sulfate, sodium chloride, potassiumchloride, calcium carbonate or the like. Inorganic elements can also beadded to a medium to adjust osmotic pressure, adjust pH, supplementtrace elements or the like. Moreover, various additives such asvitamins, nucleic acids or the like may be added to the medium forpromoting the growth of the producing strain. It is also possible to addan antifoam agent such as silicon oil, polypropylene glycol derivative,soy bean oil or the like during the culture period. A preferredtemperature range for the culture is preferably 20 to 35. degree. C.,more preferably 25 to 30.degree. C., and preferable pH of the medium is,for instance, those ranging around neutral, and the culture period is,for instance, a span of 5 to 10 days.

[0079] For recovery of the semaphorin inhibitors of the presentinvention, shown by the above-mentioned formula [17] to [37], from thefermentation broth after the cultivation, there may be adopted anyconventional methods as adopted for isolation and purification ofsecondary metabolites produced by microorganisms. These methods includesolvent extraction, ion-exchange chromatography, adsorptionchromatography, partition chromatography, gel filtration chromatography,high-performance liquid chromatography (HPLC), thin layer chromatographyand the like. These isolation and purification methods may be adoptedeither alone or in combination. To obtain the object compounds from theculture supernatant, these isolation and purification method scan beadopted. In addition, when the object compounds are within the culturedfungal mycelium, the mycelium can be collected by the means of such asfiltration or centrifugation and can be extracted directly by using awater-soluble organic solvent such as acetone, methanol or the like.Then a compound of interest can be obtained from the extract by thesimilar methods described above. Said compound of interest also can beconverted to a salt by adding an appropriate amount of base in solventssuch as water, methanol, ethanol, acetone, ethyl acetate, chloroform,ether or the like. Moreover, a hydroxyl group can be acylated, acarboxyl group can be esterificated in said compound of interest by theconventional methods. For instance, a hydroxyl group can be acylated bythe addition of an acylation agent such as acetic anhydride, acetylchloride or the like in an appropriate organic solvent and in thepresence of bases. Alternatively, a carboxyl group can be esterificatedby using alkyl halide such as methyl iodide, ethyl bromide or the likein an appropriate organic solvent and in the presence of bases. As forsaid organic solvents, acetone, ethyl acetate, chloroform, ether, DMF,pyridine or the like can be exemplified. As for bases, triethylamine,pyridine, potassium carbonate or the like can be exemplified.

[0080] Further, M162 or A721, which are described in the Examples, arealso exemplified as other semaphorin inhibitors of the presentinvention. M162 is an aliphatic compound with a weak ultra violetadsorption, whereas A721 is a natural product isolated from the culturedbroth of an actinomycete strain having a molecular weight of 437 and themaximum UV-visible absorption spectrum λ max (in methanol) at 397 nm.Considering these, M162 and A721 are low-molecular weight compoundswhose chemical structures are totally different from those of thecompounds shown by the above-mentioned formulae [17]-[37] and the like.

[0081] There is no specific limitation as to preventives or remedies ofthe present invention for neuropathic diseases and/or neurodegenerativediseases including spinal nerve injury and/or the peripheral nerveinjury, as long as they contain the nerve regeneration promotersdescribed earlier which have an inhibitor for a nerve outgrowthrepelling factor, particularly the above-mentioned semaphorininhibitors, as an active ingredient. To said preventives and remedies,various dispensing compositions may be added such as pharmaceuticallyacceptable ordinary carriers, binders, stabilizers, excipients,diluents, pH buffers, disintegrating agents, solubilizers, dissolvingcoadjuvants, isotonic agents or the like. Besides, these preventives andremedies can be administered either orally or parenterally. In otherwords, they can be administered in usual administration means, forexample, they can be orally administered in agent forms such as powder,granule, capsule, syrup, suspension liquid or the like, or they can beparenterally administered by injecting in agent forms such as solution,emulsion, suspension or the like. Alternatively, they can be nasallyadministered in the form of spray agents.

[0082] Although dosage and frequency of administration differ dependingon the method of administration and the age, weight, medical conditionsor the like of a patient, it is preferable to locally administer to thesite of disease. Since it takes several days to more than several monthsfor nerves to regenerate, the preventives or the remedies are preferablyadministered once or more than twice during that period to suppresssemaphorin activities. When administering twice or more, it ispreferable to administer the preventives or the remedies repeatedly forconsecutive days or at appropriate intervals. Dosage may be defined asseveral hundred μg to 2 g per administration in the form of a semaphorininhibitor, preferably several dozen mg or less. In order to reduce theadministration frequency, sustained release agents, an osmotic pump orthe like may be used. In any of these administration methods, it ispreferable to adopt an administration route and method wherein theconcentration should reach the sufficient level to inhibit semaphorinactivity at the site of action.

[0083] The above-mentioned neuropathic diseases and/or neurodegenerativediseases including spinal nerve injury and/or peripheral nerve injurymeans injury or degenerative diseases of peripheral or central nervesenumerated by: olfactory abnormality due to aging or the like; nerveinjury other than the olfactory caused by trauma such as spinal cordinjury or the like; nerve damage due to cerebral infarction or the like;facial nerve paralysis; diabetic neuropathy; glaucoma; retinitispigmentosa;, neurodegenerative diseases such as Alzheimer's disease,Parkinson's disease and ALS; muscular hypoplastic lateral sclerosis; LouGehrig's disease; Huntington's chorea; cerebral infarction; traumaticneurodegenerative diseases; and so on. Diseases accompanied withangiogenesis in which VEGF165 is involved are also the targets, sinceVEGF165 also uses neuropilin as its receptor.

[0084] In addition, application of the nerve regeneration promoters ofthe present invention will not be limited to pharmaceuticals such aspreventives or remedies for neuropathic diseases and/orneurodegenerative diseases, but are also capably applied to veterinarydrugs, or further to industrially important experimental reagents assemaphorin signaling inhibitors. Because they contain semaphorininhibitors as an active ingredient, nerve regeneration promoters of thepresent invention promote regeneration of olfactory nerve which is aperipheral nerve, and promote regeneration of nerves in the centralregion, which are olfactory bulb, cerebral cortex, hippocampus, corpusstriatum, thalamus, diencephalon, mesencephalon, cerebellum, pons,medulla oblongata, spinal cord, retina and the like.

[0085] The present invention is now explained in detailed with theexamples. The technical scope of the invention, however, will not belimited to these examples.

EXAMPLE 1 Production of the Compounds, SPF-3059-1, SPF-3059-2,SPF-3059-5

[0086] A 75 ml medium containing 2% glucose, 5% sucrose, 2% cotton seedpowder, 0.1% sodium nitrate, 0.1% L-histidine, 0.05% dipotassiumphosphate, 0.07% potassium chloride and 0.0014% magnesium sulfateheptahydrate, with its pH adjusted to 7.0, was pipetted to a Sakaguchiflask of 500 ml-volume and sterilized in an autoclave. A loopful ofPenicillium sp. SPF-3059 (FERM BP-7663) on slant culture was inoculatedinto this medium and cultured with shaking at 130 rpm for 5 days at27.degree. C. as the pre-culture. A medium with the same composition asthe above-mentioned medium was pipetted 300 ml each to 10 Sakaguchiflasks of 2 liter-volume and sterilized in an autoclave. Subsequently,the above mentioned pre-culture solution was added to these flasks by 6ml each, which were then cultured with shaking at 110 rmp for 7 days at27.degree. C.

[0087] After the cultivation, the fermentation broth was centrifuged at10,000 rpm for 10 minutes at 4.degree. C. to separate the supernatantand the mycelium. The supernatant fractions were extracted with a 3 L ofethyl acetate-formic acid (99:1). The mycelium fraction was extractedwith 3 liters of acetone, then filtered and concentrated. Afterconcentrated into aqueous solution, it was extracted with 1 liter ofethyl acetate-formic acid (99:1). Both extracts were then mixed andconcentrated under reduced pressure to obtain a 10.4 g of crude extract.This extract was then dissolved into 100 ml of methanol and applied to acolumn chromatography with Sephadex (trade mark) LH-20 (AmershamBiosciences K.K.), and eluted with methanol. Active fractions werecollected and the solvent was evaporated under reduced pressure toobtain 2.6 g of crude material. This crude material was then dissolvedin 100 ml of methanol for a column chromatography using TSKgel TOYOPEARLHW-40F (Tosoh Corporation) and eluted with methanol. The activefractions were collected and the solvent was evaporated under reducedpressure to obtain 1.6 g of crude substance. This crude substance wasthen dissolved in 50 mg aliquots in 1 ml of dimethyl sulfoxide (DMSO)for a reversed-phase HPLC. The conditions of the reversed-phase HPLCwere, column: Wakopak® Wakosil-II5C18RS (connecting 20×50 mm and 20×250mm, Wako Pure Chemical Industries, Ltd.), solution A: 1% aqueous formicacid solution, solution B: methanol, gradient: a linear gradient for 90min from 35% to 65% for the proportion of the solution B, flow rate: 5ml/min, detection: absorbance at 260 nm. The eluted fractions at 59, 74and 81 minutes were collected and the solvent was evaporated underreduced pressure, and thus the compounds, SPF-3059-5 (34.2 mg),SPF-3059-1 (64.1 mg) and SPF-3059-2 (12.0 mg) were obtainedrespectively. The physicochemical properties of these compounds thusobtained are as follows.

[0088] (Compound SPF-3059-1)

[0089] Appearance: yellow powder

[0090] High Resolution Fast Atom Bombardment Mass Spectrum(HRFAB-MS)m/z(M+H)⁺: Measured value: 579.0772Calculated value: 579.0776

[0091] Molecular formula: C₂₈H₁₈O₁₄

[0092] UV-VISIBLE Absorption Spectrum λ_(max) (in methanol) nm(å): 241(31,600), 315 (23,400), 365 (16,500)

[0093] Infrared Absorption Spectrum υ_(max) (KBr) cm⁻¹: 3400, 1701,1615, 1570, 1457, 1273

[0094]¹H-NMR (500 MHz, DMSO-d₆) ä ppm: 2.28, 2.67, 2.69, 4.6-4.7, 5.02,6.40, 6.91, 7.91, 8.52, 9.33, 11.1-11.6, 12.8

[0095]¹³C-NMR (125 MHz, DMSO-d₆) δ ppm: 16.5, 17.0, 32.4, 56.2, 65.7,68.0, 102.3, 104.2, 108.8, 110.1, 118.2, 118.5, 120.6, 122.2, 125.8,127.7, 132.4, 134.9, 137.6, 139.1, 140.7, 140.8, 150.1, 150.2, 152.2,153.8, 154.5, 156.3, 167.5, 167.6, 172.7, 172.8, 186.3, 199.1, 202.7,202.9

[0096] Taken together, the structure of SPF-3059-1 was determined as thefollowing formula [17] (tautomer):

[0097] (Compound SPF-3059-2)

[0098] Appearance: cream-colored powder

[0099] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M+H)⁺: Measured value: 533.0710Calculated value: 533.0721

[0100] Molecular formula: C₂₇H₁₆O₁₂

[0101] UV-VISIBLE Absorption Spectrum λ_(max) (in methanol) nm(ε): 209(40,600), 236 (42,600), 283 (28,500), 323 (25,400)

[0102] Infrared Absorption Spectrum υ_(max) (KBr) cm⁻¹: 3266, 1678,1654, 1623, 1562, 1471, 1296

[0103]¹H-NMR (DMSO-d₆) δ ppm: 2.53 (6H, s), 6.93 (1H, s), 6.95 (1H, s),7.47 (1H, s), 8.15 (1H, s), 8.54 (1H, s), 9.38 (1H, brs), 9.89 (1H,brs), 10.78 (1H, brs), 11.37 (1H, brs), 12.68 (1H, brs)

[0104]¹³C-NMR (DMSO-d₆) δ ppm: 29.1, 32.1, 102.3, 103.1, 108.7, 112.5,113.5, 119.6, 119.8, 120.9, 126.2, 132.4, 133.6, 136.1, 141.7, 144.5,150.71, 150.74, 152.49, 152.54, 152.7, 154.4, 167.4, 172.9, 173.4,199.2, 201.2

[0105] Taken together, the structure of SPF-3059-2 was determined as thefollowing formula [18]:

[0106] (Compound SPF-3059-5)

[0107] Appearance: cream-colored powder

[0108] High Resolution Fast Atom Bombardment Mass Spectrum(HRFAB-MS)m/z(M+H)⁺: Measured value: 577.0615Calculated value: 577.0619

[0109] Molecular formula: C₂₈H₁₆O₁₄

[0110] UV-VISIBLE Absorption Spectrum λ_(max) (in methanol) nm(ε): 229(35,800), 284 (22,600), 322 (21,000)

[0111] Infrared Absorption Spectrum υ_(max) (KBr) cm⁻¹: 3260, 1684,1626, 1567, 1467, 1288

[0112]¹H-NMR (DMSO-d₆) δ ppm:

[0113] 2.53 (3H, s), 2.55 (3H, s), 6.93 (1H, s), 6.96 (1H, s), 8.17 (1H,s), 8.53 (1H, s), 9.5-13.0 (6H)

[0114]¹³C-NMR (DMSO-d₆) δ ppm: 29.1, 32.1, 102.26, 102.32, 109.9, 112.4,119.6, 119.8, 120.3, 120.9, 126.3, 132.5, 133.4, 136.2, 141.2, 141.7,150.4, 150.8, 152.1, 152.68, 152.73, 154.5, 167.4, 167.5, 172.5, 172.9,199.1, 201.1

[0115] From these results, the structure of SPF-3059-5 was determined asthe following formula [19]:

EXAMPLE 2 Suppressing Action of the Compounds SPF-3059-1, SPF-3059-2,SPF-3059-5, M162 and A721 of the Present Invention to the CollapseActivity of Sema3A

[0116] A 96-well plate (Sumitomo Bakelite Co. ,ltd.) pre-coated withpolylysine was further coated with laminine (20 μg/ml of laminine, for 1hour at room temperature). Each well was added with 100 μl of medium(F12 medium containing 10% bovine fetal serum, 20 ng/ml of NGF, 100units/ml of penicillin and 100 μg/ml of streptomycin) which media werethen inoculated with dorsal root nerve ganglions excised from E7(Embryonic day 7) chick embryo and were cultured for 16 to 20 hoursunder 5% CO₂ and at 37. degree. C. Subsequently, the object compoundswere added to media at various concentrations and 2 units/ml of mousesemaphorin 3A (Sema3A) was added after cultivating for 1 hour. Thecultures were further incubated for another 1 hour. Glutaraldehyde wasquickly added therein after said 1 hour to make the final concentration1%. The cultures were then left for 15 minutes at room temperature sothat the tissue sections were fixed, and collapse rates of the growthcones were microscopically observed. A well with no addition of Sema3Awas made the control. The results are shown in FIGS. 1 and 2.

[0117]FIGS. 1 and 2 show that the collapse rate of the growth conesdecrease as the compound concentration increase. On the contrary,compounds alone did not affect the rates at all. The results revealedthat these compounds (SPF-3059-1, SPF-3059-2, SPF-3059-5, M162 and A721)inhibit the growth cone collapse activities of Sema3A in aconcentration-dependent manner. The longitudinal and lateral axes in thefigures represent the collapse rate of growth cones and theconcentration of compounds, respectively.

show the results when Sema3A was added after the addition of thecompounds and ◯□Δ show those when Sema3A was not added. Further, IC50(μg/ml) of the inhibitors were determined by the method comprising:calculating the collapse rate of growth cone (A)% of the negativecontrols (neither compounds nor Sema3a was added); subsequentlycalculate the collapse rate of growth cone (B)% of the positive controls(compounds were not added but Sema3a was added); and the concentrationwas chosen for each compound from the chart where the collapse rate ofgrowth cone met (A+B)/2 (%), which was made IC50 level. The followingsare the results. Compound IC50 (μg/ml) SPF-3059-1 <0.1 SPF-3059-2 <0.1SPF-3059-5 <0.1 M162 2.0 A721 5.0

[0118] These results show that SPF-3059-1, SPF-3059-2 and SPF-3059-5potently inhibit semaphorin.

EXAMPLE 3 Suppressing Action of SPF-3059-1 to the Collapse Activity ofSema6C

[0119] The experiment was carried out in a same way as in Example 2except that rat semaphorin 6C-AP (Sema6C-AP: a fusion protein ofextracellular domain of Sema6C and human placenta-derived alkalinephosphatase) was used instead of mouse semaphorin 3A (Sema3A), anddorsal root ganglions excised from 8-day-old chick embryos were usedinstead of those excised from 7-day-old chick embryos. The collapse rateof growth cone was measured when Sema6C-AP was added one hour after theaddition of the compound SPF-3059-1 at various concentrations. Theresults are shown in FIG. 3. The longitudinal and lateral axes in thefigure represent the collapse rate of growth cones and the concentrationof SPF-3059-1, respectively.

shows the result when Sema6C-AP was added after the addition ofSPF-3059-1 and shows the result when Sema6C-AP was not added. FIGS. 3shows that the collapse rate of the growth cones decrease as theSPF-3059-1 concentration increased. On the contrary, SPF-3059-1 alonedid not affect the rate at all. These results revealed that SPF-3059-1inhibit the growth cone collapse activities of Sema6C-AP in aconcentration-dependent manner.

EXAMPLE 4 Suppression of Neurite Outgrowth Inhibitory Action of Sema3Aby the Inhibitors

[0120] Whether the object compounds (SPF-3059-1, M162, A721) havepersistent inhibitory action to Sema3A was analyzed by the collagen gelco-culture method (Neuroprotocols 4, 116, 1994) using Sema3A-expressingCOS7 cell clumps and 7- or 8-day-old chick embryo dorsal root ganglions.The Sema3A-expressing COS7 cell clumps were generated as follows. 1 μgof Sema3A-expression plasmid was introduced into COS7 cells (100000cells/35 mm culture plate), which were cultured overnight, with theFuGENE6 transfection reagent (Roche). 2.5 hours after the starting ofthe transfection, the COS7 cells were collected by trypsinization andcentrifugation, and re-suspended in a medium of 200 μl. 20 μl of thecell suspension was placed on the lid of the culture plate (inside) andthe lid was turned over, and then the suspension was cultivated for 20hours (hanging drop culture) (Cell 78, 425, 1994). When the culture wasover, aggregated COS7 cells (clump) were collected and trimmed to adiameter of 0.5 mm. The Sema3A-expressing COS7 cell clump and theabove-mentioned dorsal root ganglions were placed in parallel at adistance of 0.5 to 1 mm in a 0.2% collagen gel which was then cultivatedin a medium containing the aforementioned compounds at variousconcentrations for 2 days at 37. degree. C. under 5% CO₂. Glutaraldehydewas quickly added therein to make the final concentration 1%. Thecultures were then left for 1 hour at room temperature so that thetissues were fixed, and the neurite outgrowth was microscopicallyobserved. The results are found in FIG. 4.

[0121] The concentration gradients were formed in the above-mentionedcollagen gels because Sema3A was secreted from the COS7 cell clump whichwas introduced with the Sema3A-expressing plasmids (The one nearer tothe COS7 cell clump had a higher concentration). When a medium notcontaining a subject compound was used, neurites were unable to growtowards the COS7 cell clump with the high concentration of Sema3A andgrew only to the opposite direction. However, when the compoundSPF-3059-1 or M162 was added to the medium, neurite outgrowth wasobserved to the direction toward the Sema3A-expressing COS7 cell clump.Such neurite outgrowth toward the Sema3A-expressing COS7 cell clump wasmore remarkable as the compound concentration was higher, suggesting theconcentration-dependency. This result showed the ability of SPF-3059-1and M162 to persistently inhibit Sema3A activities. However, only whenA721 was added, neurite outgrowth toward the Sema3A-expressing COS7 cellclump, which was observed with the addition of SPF-3059-1 and M162, wasnot observed. This showed that A721 did not have the persistinginhibitory action on Sema3A.

EXAMPLE 5 Suppression of Neurite Outgrowth Inhibitory Action of Sema3A

[0122] In a similar way as in Example 4, suppression of the neuriteoutgrowth inhibitory activity was analyzed for the compounds,SPF-3059-1, SPF-3059-2 and SPF-3059-5. When the neurites outgrew in acomplete concentric circular shape just like for the control groupswhere Sema3A-non-expressing COS7 cells were used, it was scored +++ (astrong inhibitory effect for Sema3A). When the outgrowth was foundalmost in a concentric circular shape accompanied with a littlesuppression of the outgrowth toward the direction of Sema3A-expressingCOS cells, it was scored ++. When the outgrowth toward the direction ofSema3A-expressing COS cells is fairly suppressed to present a crescentshape, it was scored +. When the outgrowth toward the direction ofSema3A-expressing COS cells was not found at all (no inhibitory effectfor Sema3A), it was scored −. The determined results are shown below.Concentration of the subject compounds (μg/ml) Compound 0.5 1.0 2.0SPF-3059-1 + ++ +++ SPF-3059-2 + ++ +++ SPF-3059-5 + ++ +++ PBS(control) − − −

[0123] These results revealed that the compounds, SPF-3059-1, SPF-3059-2and SPF-3059-5 persistently inhibited the action of Sema3A secreted fromthe Sema3A-expressing COS7 cells during the 48-hour culture.

EXAMPLE 6 In vivo Nerve-Regeneration Promoting Action of the SemaphorinInhibitor SPF-3059-1 in the Olfactory Nerve Axotomy

[0124] Male Wister rats (6.5-week-old) were purchased from CHARLES RIVERJAPAN, INC. and bred under free feeding and water uptake in thededicated breeding room. A sample solution was prepared by dilutingSPF-3059-1 to 1 mg/ml with PBS and filled in an osmotic pressure pump(alzet 2004, ALZA co., USA). A pump filled with PBS was used as acontrol. The sample solution and the like were prepared on the daybefore the operation and the osmotic pressure pump was bathed in PBS andplaced overnight at room temperature. Just before the operation, aL-cannula was connected to the one end of a silicon tube filled with thesample solution and the like, and the other end of the tube wasconnected to the pump. A rat was anesthetized with pentbarbital (50mg/kg, intrapenitrially injected), and its head was fixed to thestereotaxic apparatus. The scalp was incised along the median and thecranium above the olfactory bulb was opened to expose the olfactory bulb(anterior part). In order to axotomize olfactory nerve, a knife (a razorwas cut into 1.5 mm width) was inserted in between the olfactory bulband the cribriform plate. The olfactory nerve which is projected on theupper olfactory bulb is axotomized by this handling. The L-cannula wasfixed to the cranium-opening above the olfactory with surgical instantadhesive and dental cement so that the edge of cannula is located in thevicinity of the incision. The sample output was set to 6 μl (6 μg)/day.The pump was subcutaneously inserted in the dorsal neck and the incisionwas sutured. Then the animal was recovered.

[0125] Two and three weeks after the operation, the rat anesthetizedwith pentbarbital was laid on its back and injected with 100 μl of 1%WGA-HRP/PBS (TOYOBO) into the nasal cavity using a microsyringe. 24hours after the HRP injection, the rat was further anesthetized withpentbarbital (50 mg/kg, intrapenitrially injected) and was thoracicallyincised. Subsequently, PBS was perfused from the left ventricle and thenPBS containing 200 ml of 4% paraformaldehyde was perfused. The olfactorybulb was excised and placed in PBS containing 30% sucrose, was bathedtherein overnight at 4. degree. C., and was then frozen up with dry ice.The olfactory bulb was embedded in an OTC compound on dry ice and then30 μm sections were prepared with a cryostat. The sections werealiquoted in Tris-buffered saline (TBS) (one in every three pieces). Thesections were washed in TBS where a 0.1 M TBS containing 0.48% DAB,0.096% NiCl and 0.036% H₂O was then added and reaction took place for 15min. After having been washed in TBS, the sections were mounted on aglass slide and sealed after they were dried. The sections weremicroscopically observed and HRP reaction of the glomerulus at the outerportion of olfactory bulb was observed.

[0126] Regeneration of olfactory nerves was quantitatively assessed byobserving in which horizontal section of the olfactory bulb from thevertex did HRP-positive glomerulus emerge. The results are shown in FIG.5 and Table 1. All the rats except the one which died from suffocationon Week 2 at the HRP injection were used for the assessment. WhenSPF-3059-1 was injected, HRP-positive sections were found in shallowerpieces than the controls injected with PBS at both Weeks 2 and 3. Thesignificant differences were judged in the SPF-3059-1-injected groupagainst the PBS-injected group, the HRP-positives were found in theshallower sections than the PBS-injected group at Week 3. These resultsshow that SPF-3059-1 has marked nerve-regeneration promoting action inthe adult rat models for olfactory nerve axotomy. TABLE 1 Positivesection No. (which section from the Number of Agent vertex, average ±SE) rats Week 2 PBS 11.8 ± 0.3  4 Week 2 SPF3059-1 8.3 ± 1.9 3 Week 3PBS 8.0 ± 1.4 4 Week 3 SPF3059-1  5.2 ± 1.9* 5

EXAMPLE 7 In vivo Nerve-Regeneration Promoting Action of the SemaphorinInhibitors, M162 and A721 in the Olfactory Nerve Axotomy

[0127] The same experiment as in Example 6 was carried out with M162 andA721. The results are shown in FIG. 6. These results show that M162 hasthe in vivo nerve-regeneration promoting action but A721 does not.

EXAMPLE 8 In vivo Nerve-Regeneration Promoting Action of the SemaphorinInhibitor SPF-3059-1 in the Sciatic Nerve Crush

[0128] Male Wister rats were purchased at 7-week-old (CHARLES RIVERJAPAN, INC.) and bred under the light and dark cycles for 12 hours. Therats had free access to solid feedstuff (CLEA Japan, Inc., CE2) andwater, and were used in the experiment after about one week ofpreliminary breeding. Under pentbarbital anesthesia, sciatic nerve ofthe rat thighs was exposed, clipped for 30 sec with forceps of 5 mmwidth, and was thus crushed. The crush site was marked with 10-0 nylonthread. SPF-3059-1 was dissolved in PBS at 8.3 ug/ml and was locallyadministered to the lesion site for 14 consecutive days at a flow rateof 1 ug/day by using an osmotic pressure pump (2ML2, 5 ul/hr, alzet).PBS was administered to the controls. At Day 14 of administration, thesciatic nerves were excised and trimmed at the distal areas from thecrush site (the positions of marking) by 2 mm, 6 mm and 10 mm. Theincised sciatic nerves were fixed in 2.5% glutaraldehyde/0.1 M phosphatebuffer for a day and a night. When they were fixed, nerves weredehydrated by alcohol and were embedded in epoxy resin. The transversesections of the sciatic nerves were made and dyed with toluidine-blue,and their photographs were taken under an optical microscope todetermine the counts of myelinated fibers. After the areas of sciaticnerves were measured, the myelinated fiber density was calculated bydividing each area by the count of myelinated fibers. The results areshown in FIG. 7. The myelinated fiber density was increased more in theSPF-3059-1-injected group compared to that of the PBS-injected group atthe distal areas of 2 mm and 6 mm from the crush site. This result showsthat SPF-3059-1 has the promoting effect for the regeneration of sciaticnerves that were crushed.

EXAMPLE 9 Effect of the Semaphorin Inhibitor SPF-3059-1 On CellProliferation

[0129] COS7 cells were seeded onto a 96-well plate (medium 100 μl/well)at 10000 cells per well. At the same time, SPF-3059-1 of variousconcentrations was added and the cells were cultivated for two days inthe presence of 5% CO₂ at 37.degree. C. Each well was then added with 10μl of MTT solution at 5 mg/ml and cultured for another one hour.Subsequently, the culture supernatant was removed and formazan (an MTTderivative produced inviable cells) accumulated in the cells wasdissolved by the addition of 50 μl DMSO and then the absorbance at 570nm was measured to determine the proliferation of COS7 cells. Theresults are shown in FIG. 8. As shown in FIG. 8, the level of cellproliferation in the culture carried out in the presence of SPF-3059-1was similar to the level observed in the absence of SPF-3059-1. Thisresult shows that SPF-3059-1 has no suppressing activity for cellproliferation. The longitudinal and lateral axes in the figure show cellproliferation and the SPF-3059-1 concentration, respectively. Besides,the standard deviation (n=4) was added to each column.

EXAMPLE 10 (Effects of the Semaphorin Inhibitors SPF-3059-1, SPF-3059-2and SPF-3059-5 on Cell Proliferation

[0130] The same effect on cell proliferation as in Example 9 wasexamined for the compounds, SPF-3059-1, SPF-3059-2 and SPF-3059-5. Thewells with these compounds were served as samples, the wells with PBSwere served as control, and the wells for which the experiment wascarried out without adding the cells was served as blank. IC50 (μg/ml),the inhibitory rate for cell proliferation, was determined, which iscalculated by the equation shown below.

[0131] The inhibitory rate for cell proliferation (%)=(1−(absorbance ofa sample well−absorbance of the blank well)/(absorbance of the controlwell−absorbance of the blank well))×100

[0132] The determination results of the inhibitory rate for cellproliferation of the above-mentioned compounds are as follows, showingthat no cytotoxicity was observed at 1000 to 3000 folds or even higherconcentrations in which semaphorin inhibitory activities can beobserved. Compound IC50 (μg/ml) SPF-3059-1 >300 SPF-3059-2 >100SPF-3059-5 >300

EXAMPLE 11 Inhibitory Activity of Sema3A-Receptor-Binding

[0133] Whether SPF-3059-1 inhibits the binding of Sema3A andNeuropilin-1, which is a component constituting Sema3A receptor complex,was examined in the receptor-binding experiment. Neuropilin-1 and plexinA1 are currently known as constituting components of Sema3A receptors.However, mainly Neuropilin-1 is known to contribute to Sema3A binding.Sema3A fused with alkaline phosphatase (human-derived: heat-resistance)(Sema3A-AP) was used as a ligand in the receptor-binding experiment andthe amount bound to the receptor was detected with the alkalinephosphatase activity as an index. Mouse-derived Sema3A was used. Arecombinant gene was generated in which alkaline phosphatase was fusedfrom the 758th amino acid to the C terminal side in said Sema3A. Thisrecombinant gene was then introduced into a COS7 cell where it wasexpressed and secreted and Sema3A-AP was prepared.

[0134] The receptor-binding experiment was performed as described below.The Neuropilin-1 expressing plasmid (pUCSRα-Neuropilin-1) was introducedinto COS7 cells and cultivated for 24 hours. These cells expressNeuropilin-1 on the cell surface. The cells were washed once in HBHbuffer solution (Hank's balanced salt solution containing 20 mM HEPES,pH 7.2, and 0.5 mg/ml bovine serum albumin) and then HBH buffer solutioncontaining Sema3A-AP, and SPF-3059-1 of various concentrations (0 to 10μg/ml) were added at the same time. After having been left for one hourat room temperature while being shaken, Sema3A-AP was bound toNeuropilin-1 expressing cells, after which the supernatant was removed.Subsequently, the cells were washed six times in HBH buffer solution toremove the excess Sema3A-AP. Thereafter Sema3A-AP bound to the cells wassolubilized with 10 mM Tris-HCl, pH 8.0, 1% Triton X-100 solution (thecell extract). Insoluble materials in the cell extract was removed bycentrifugation and then endogenous alkaline phosphatase of the cell perse was inactivated after the treatment for one hour at 65.degree. C. Thealkaline phosphatase activity derived from Sema3A-AP in the cell extract(=binding amount of Sema3A-AP) was determined. An aliquote of the cellextract was admixed with SEAP buffer (1M diethanolamine, 0.5 mM MgCl₂ 10mM L-homoarginine) and a fluorescent substrate (10 mM p-nitirophenylphosphate), and kept warm at 37.degree. C. The solution was thenmeasured for its absorbance at 405 nm (p-nitrophenol: generated fromp-nitirophenyl phosphate with alkaline phosphatase). The results areshown in FIG. 9. The results show that the binding of Sema3A-AP decreasein a concentration-dependent manner with increasing concentration ofSPF-3059-1. This clearly shows that SPF-3059-1 inhibits the Sema3Aactivity because the compound inhibits the binding of Sema3A to itsreceptor.

EXAMPLE 12 (Inhibition of Collapse Activity by the Contact of Sema3A andthe Inhibitor

[0135] Sema3A was mixed with SPF-3059-1, which concentration was highenough to show the inhibitory activity (0.25 μg/ml) (=Pre-Mix sample).Thereafter the Pre-Mix sample was added to the culture solution ofdorsal root ganglions to examine whether the growth cone collapseactivity of Sema3A was inhibited. The amount of Pre-Mix sample was aquarter of that of the culture solution. This means that the SPF-3059-1concentration will be diluted from 0.25 μg/ml to 0.05 μg/ml (1/5 fold)when the Pre-Mix sample is added to the culture solution of dorsal rootganglions. This is the concentration where the inhibitory activity willnot be observed when SPF-3059-1 and Sema3A are independently added. Theresults are shown in FIG. 10. FIG. 10 shows that Sema3A activity wasinhibited when the Pre-Mix sample was added to the culture solution ofdorsal root ganglions in spite of the fact that the final concentrationof SPF-3059-1 was 0.05 μg/ml (concentration of no-inhibitory activity).This means that the activity of Sema3A will be lost at the moment whenSema3A and SPF-3059-1 come in contact. From this result, the targetmolecule of SPF-3059-1 is thought to be Sema3A.

EXAMPLE 13 In vivo Nerve-Regeneration Promoting Action of the SemaphorinInhibitor SPF-3059-1 in the Transaction of the Spinal Nerve (CerebralCortex-Spinal Cord Tract)

[0136] Male Wister rats (10-week-old) were purchased from Charles RiverJapan, Inc. and bred under free feeding and water uptake in a dedicatedbreeding room. They were used in the experiment after about one week ofpre-breeding. SPF-3059-1 was prepared to 0.1 mg/ml with PBS. PBS wasused as a control. The agent was filled in an osmotic pressure pump(Alzet, model 2004, dosage for four weeks, flow rate 0.25 μl/hr). Acannula filled with the sample solution etc. was connected to the pumpand pre-incubated in saline overnight at room temperature.

[0137] A rat was intrapenitrially injected with pentbarbital (50 mg/kg).Skin and muscles in dorsal thoracic spinal cord region of theanesthetized rat were incised and the T8-T12 vertebras were exposed.Laminectomy was carried out for the T11 thoracic vertebra under themicroscope. A pair of ophthalmologic scissors attached to themanipulator was punctured and inserted across the median line into thedepth of 1.5 mm from the duramater surface, and the cerebralcortex-spinal cord pathway was incised and separated. The vertebraopening was filled with a spongel. Cannulation was performed from the T9vertebra which was perforated with a surgical drill to expose theduramater. The part of the duramater, to which a cannula was to beinserted, was perforated with an injection needle and the silicon tubeconnected with the pump was inserted therein. It was made sure that theedge of the cannula reached to the T11 spinal cord injured area. Then aspongel was filled in the vertebra opening where a drop of surgicalinstant adhesive was infiltrated. The cannula was fixed to the adjacentmuscle with suture to prevent slipping off. After suturing the muscleoperated, the incised skin was stitched together with suture clamps. Thesample was administered for four consecutive weeks with the sample flowrate of 0.6 μg/day.

[0138] Two weeks after the operation, the rat was anesthetized withpentbarbital (50 mg/kg, intrapenitrially injected) whose head was fixedto the stereotaxic apparatus. The scalp was incised along the medianline and the cranium was perforated with a drill to expose the cerebrum.10% DBA (Dextran Biotinylated Amine) was injected 0.1 μl each to 18spots in the cerebrum cortex motor area with a microsyringe, after whichthe incision was sutured and the animal was recovered. DBA will betransported from the nerve nucleus to the spinal cord pathway nerves.After two weeks of breeding, the rat was anesthetized again withpentbarbital and its thoracic part was incised and then PBS was perfusedfrom the left ventricle. Subsequently, PBS containing 200 ml of 4%paraformaldehyde was perfused. The spinal cord (including lesioned part)was taken out and fixed overnight in the solution, which was then placedin PBS containing 30% sucrose and bathed therein at 4.degree. C. Thespinal cord was embedded in the OTC compound and frozen on dry ice. 30μm sections were prepared with a cryostat and aliquoted in Tris buffersolution (TBS). The sections were washed in TBS and bathed in ABCreaction solution for two hours. After the sections were washed, theywere visualized with DBA substrate and the preparations were made.Regenerated nerve fibers in the lesioned area were microscopicallyobserved. FIG. 11 shows the result of the injection of SPF-3059-1 of thepresent invention and FIG. 12 shows the result of PBS injection as acontrol. FIG. 11A, B and C and FIG. 12A, B and C show serial sections inthe proximal of traumatized region, respectively. FIG. 11D and FIG. 12Dare large macrographs of FIG. 11B and FIG. 12B, respectively. WhenSPF-3059-1 was administered, the retention of DBA (dark arrowhead) wasobserved in the rostral part to the lesion site as shown in FIG. 11D.Many DBA-positive fibers were observed extending from the retention areato the caudal along the lesioned area (light arrowhead) and regeneratednerve fibers that extended evading the incision site were frequentlyobserved. On the contrary, when PBS was administered, DBA retention(dark arrowhead) was observed in the rostral part to the lesion site asshown in FIG. 12D. A few DBA-positive fibers were observed to the caudalside along the lesioned area (light arrowhead) and only a few nervefibers were observed. These results revealed that SPF-3059-1 has thenerve-regeneration promoting action in the spinal cord injury modeladult rats.

EXAMPLE 14 Production of the Novel Compounds of the Present Invention

[0139] A 10 ml medium containing 2% glucose, 5% sucrose, 2% cotton seedpowder, 0.1% sodium nitrate, 0.1% L-histidine, 0.05% dipotassiumphosphate, 0.07% potassium chloride and 0.0014% magnesium sulfateheptahydrate, with its pH adjusted to 7.0, was pipetted to a Erlenmeyerflask of 50 ml volume and sterilized in an autoclave. A loopfulPenicillium sp. SPF-3059 (FERM BP-7663) on slant culture was inoculatedinto this medium and cultured with shaking at 180 rpm at 27° C. for 4 aspre-culture. A medium with the same composition as the above-mentionedmedium was pipetted 125 ml each to five Erlenmeyer flasks of 500 mlvolume and sterilized in an autoclave. Subsequently, the above-mentionedpre-culture solution was added 4 ml each to the five flasks and culturedwith shaking at 180 rpm for 4 days at 27.degree. C. A 30 liters ofmedium containing 1.43% glucose, 3.57% sucrose, 1.43% cotton seedpowder, 0.07% sodium nitrate, 0.07% L-histidine, 0.036% dipotassiumphosphate, 0.05% potassium chloride, 0.001% magnesium sulfateheptahydrate and 0.01% Adekanol LG-295S (antiforming agent by AsahiDenka Co.,Ltd), with its pH adjusted to 7.0, in a jar fermentor of 50liters volume was sterilized under high-pressure steam (121.degree. C.,20 min). Then 500 ml of the above-mentioned cultured broth was added tothe fermentor and cultured at 27.degree. C. for 9 days with an agitationof 400 rpm and an aeration of 15 liters/min.

[0140] When the culture was finished, the cultured broth was centrifugedfor 10 minutes at 10,000 rpm to separate the supernatant and themycelium. The supernatant fraction was extracted twice with a 20 litersof ethyl acetate-formic acid (99:1). The fungus mycelium fraction wasextracted with 30 liters of acetone, and then filtered and concentrated.After concentrated into aqueous solution, it was extracted with 10liters of ethyl acetate-formic acid (99:1). Both extracts were thenmixed and concentrated under reduced pressure to obtain 224 g of crudeextract. 100 g of this crude extract was dissolved in 500 ml of methanoland applied to a column chromatography using Sephadex (trademark) LH-20(Amersham Biosciences K. K.), and eluted with methanol. Active fractionswere collected and the solvent was evaporated under reduced pressure toobtain 48.8 g of oily substance. The substance was then dissolved in 400ml of methanol and applied to a column chromatography using TSKgelTOYOPEARL HW-40F (Tosoh Corporation), and eluted with methanol. Theactive fractions were collected and the solvent was evaporated underreduced pressure to obtain 21.8 g of crude substance. This crudesubstance was then dissolved in 200 mg aliquots in 2 ml of DMSO andapplied to a preparative reversed-phase HPLC. The conditions of thepreparative reversed-phase HPLC were, column: Wakopak®Wakosil-II5C18HGprep (connecting 5 i.d.×10 cm and 5 i.d.×25 cm, WakoPure Chemical Industries, Ltd.), solution A: 1% aqueous formic acidsolution, solution B: methanol, gradient: a linear gradient for twohours from 45% to 75% for the proportion of the solution B, flow rate:25 ml/min, and detection: absorbance at 260 nm. The eluate wasfractioned by one minute.

[0141] The eluted fractions as described above were analyzed byanalytical HPLC. The conditions of the analytical HPLC were, column:Wakopak® Wakosil-II5C18RS (4.6×150 mm, Wako Pure Chemical Industries,Ltd.), solution A: 1% aqueous formic acid solution, solution B:methanol, gradient: a linear gradient for 71.1 min from 20% to 67% forthe proportion of the solution B, flow rate: 1.3 ml/min, and detection:absorbance at 260 nm. The fractions containing the object compound werecollected with the retention time in this analytical HPLC as the index,and the solvent was evaporated under reduced pressure. The resultingmaterial was again applied to the preparative HPLC and purified in asimilar manner as in the above, and were further applied to a columnchromatography using TSKgel TOYOPEARL HW-40F (Tosoh Corporation) andpurified similarly as in the above. Fractions containing the objectcompound were collected and the solvent was evaporated under reducedpressure. Thereby, the purified compounds described below were obtained.Retention time in Amount the analytical HPLC Compound obtained (mg)(min) SPF-3059-12 6.2 34.4 SPF-3059-24 28.0 34.5 SPF-3059-4 10.2 36.0SPF-3059-25 4.0 39.3 SPF-3059-34 2.9 40.8 SPF-3059-6 34.9 45.6SPF-3059-27 17.4 46.1 SPF-3059-26 6.2 46.5 SPF-3059-28 11.8 46.6SPF-3059-7 13.0 47.0 SPF-3059-39 2.8 49.95 SPF-3059-37 4.0 50.0SPF-3059-3 118.2 50.1 SPF-3059-35 11.0 51.5 SPF-3059-9 100.9 52.7SPF-3059-29 45.6 54.0 SPF-3059-36 3.7 57.8 SPF-3059-30 23.5 63.0

[0142] Physicochemical properties of the compounds obtained are asfollows:

[0143] (SPF-3059-3)

[0144] Appearance: yellow powder

[0145] Molecular weight: 534

[0146] Molecular formula: C₂₇H₁₈O₁₂

[0147] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 535(M+H)⁺

[0148] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 533(M−H)⁺

[0149] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M+H)⁺: Measured value: 535.0905

[0150] Calculated value: 535.0877 (C₂₇H₁₉O₁₂)

[0151] UV-VISIBLE Absorption Spectrum λ_(max) (in methanol) nm (ε): 242(30,800), 317 (22,700), 367 (14,000)

[0152] Infrared Absorption Spectrum υ_(max) (KBr) cm⁻¹: 3356, 1700,1652, 1610, 1515, 1475, 1283

[0153]¹H-NMR (DMSO-d₆) δ ppm: 2.28, 2.29, 2.68, 2.69, 4.62, 4.62, 4.64,4.72, 5.03, 6.38, 6.40, 6.90, 6.91, 7.44, 7.98, 8.54, 8.90-11.10, 12.70

[0154]¹³C-NMR (DMSO-d₆) δ ppm: 16.5, 17.0, 32.3, 32.4, 56.2, 65.8, 68.0,103.0, 104.2, 108.7, 108.8, 109.4, 113.5, 118.2, 118.6, 122.2, 125.7,127.5, 129.8, 132.0, 132.6, 134.8, 137.6, 137.9, 138.8, 144.3, 150.5,150.6, 152.0, 152.6, 154.2, 154.5, 155.5, 156.3, 167.6, 167.7, 173.4,183.5, 186.2, 199.2, 202.8, 203.0

[0155] Solubility:

[0156] Insoluble: water, hexane Soluble: methanol, DMSO

[0157] Taken together, the structure of SPF-3059-3 was determined as thefollowing formula [20] (tautomer).

[0158] (SPF-3059-4)

[0159] Appearance: Cream-colored powder

[0160] Molecular weight: 560

[0161] Molecular formula: C₂₈H₁₆O₁₃

[0162] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 561(M+H)⁺

[0163] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 559(M−H)⁻

[0164] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z (M+H)⁺: Measured value: 561.0667Calculated value: 561.0670(C₂₈H₁₇O₁₃)

[0165] UV-VISIBLE Absorption Spectrum λ_(max) (in methanol) nm (C): 221(35,600), 250 (38,100), 276sh (25,800), 323 (24,300)

[0166] Infrared Absorption Spectrum υ_(max) (KBr) cm⁻¹: 3412, 1665,1619, 1563, 1465, 1427, 1263

[0167]¹H-NMR (DMSO-d₆) δ ppm: 2.53 (3H, s), 2.56 (3H, s), 6.84 (1H, d,2.1), 6.95 (1H, s), 6.96 (1H, d, 2.1), 8.17 (1H, s), 8.52 (1H, s),10.10-11.40 (3H, brs), 12.71 (1H, brs), 13.26 (1H, brs)

[0168]¹³C-NMR (DMSO-d₆) δ ppm: 29.2, 32.1, 102.3, 103.2, 110.1, 112.4,112.8, 119.6, 120.3, 120.8, 126.3, 133.1, 133.4, 136.7, 137.5, 141.7,150.8, 152.3, 152.7, 152.8, 157.2, 163.9, 167.4, 169.3, 172.2, 172.9,199.3, 201.0

[0169] Solubility:

[0170] Insoluble: water, hexane Soluble: methanol, DMSO

[0171] Taken together, the structure of SPF-3059-4 was determined as thefollowing formula [21].

[0172] (SPF-3059-6)

[0173] Appearance: Cream-colored powder

[0174] Molecular weight: 592

[0175] Molecular formula: C₂₉H₂₀O₁₄

[0176] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 593(M+H)⁺

[0177] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 591(M−H)⁻

[0178] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M+H)⁺: Measured value: 593.0949Calculated value: 593.0932(C₂₉H₂₁O₁₄)

[0179] UV-VISIBLE Absorption Spectrum λ_(max) (in methanol) nm (ε):210sh (45,900), 223 (47,700), 317 (25,800), 358sh (14,700)

[0180] Infrared Absorption Spectrum υ_(max) (KBr) cm¹: 3418, 1701, 1617,1565, 1465, 1301

[0181]¹H-NMR (DMSO-d₆) δ ppm: 2.22 (3H, s), 2.72 (3H, s), 3.11 (3H, s),3.98 (2H, brs), 6.78 (1H, s), 6.88 (1H, s), 8.21 (1H, s), 9.00-13.00(6H, brs)

[0182]¹³C-NMR (DMSO-d₆) δ ppm: 16.8, 32.4, 57.8, 64.4, 102.1, 102.3,109.3, 111.9, 118.4, 118.6, 119.1, 119.6, 127.6, 128.2, 131.6, 139.0,141.6, 142.1, 150.7 (2C), 151.9, 152.9, 155.5, 162.0, 167.8, 167.9,172.4, 174.6, 202.6

[0183] Solubility: Insoluble: water, hexane Soluble: methanol, DMSO

[0184] Taken together, the structure of SPF-3059-6 was determined as thefollowing formula [22].

[0185] (SPF-3059-7)

[0186] Appearance: Yellow powder

[0187] Molecular weight: 562

[0188] Molecular formula: C₂₈H₁₈O₁₃

[0189] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 563(M+H)⁺

[0190] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 561(M−H)⁻

[0191] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M+H)⁺: Measured value: 563.0843Calculated value: 563.0826 (C₂₈H₁₉O₃)

[0192] UV-VISIBLE Absorption Spectrum λ_(max) (in methanol) nm (ε): 242(31,600), 312 (24,500), 385 (10,200)

[0193] Infrared Absorption Spectrum υ_(max) (KBr) cm⁻¹: 3424, 1701,1603, 1504, 1448, 1420, 1270

[0194]¹H-NMR (DMSO-d₆) δ ppm: 2.28, 2.29, 2.68, 2.69, 4.62, 4.67, 4.71,5.04, 6.38, 6.41, 6.81, 6.92, 6.93, 7.95, 8.52, 9.33, 11.22, 11.35,12.93

[0195]¹³C-NMR (DMSO-d₆) δ ppm: 16.6, 17.0, 32.3, 32.4, 56.2, 65.7, 67.9,102.3, 102.8, 103.1, 104.3, 108.7, 109.3, 110.1, 112.5, 112.6, 118.5,118.7, 121.6, 122.1, 125.9, 127.6, 130.1, 131.9, 132.4, 135.3, 137.4,137.6, 138.9, 139.7, 151.9, 152.3, 154.5, 155.5, 156.2, 157.1, 163.6,167.6, 169.3, 172.7, 172.8, 183.7, 186.2, 199.1, 202.5, 202.7

[0196] Solubility: Insoluble: water, hexane Soluble: methanol, DMSO

[0197] Taken together, the structure of SPF-3059-7 was determined as thefollowing formula [23] (tautomer).

[0198] (SPF-3059-9)

[0199] Appearance: Yellow powder

[0200] Molecular weight: 534

[0201] Molecular formula: C₂₇H₁₈O₁₂

[0202] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 535(M+H)⁺

[0203] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 533(M−H)⁻

[0204] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M+H)⁺: Measured value: 535.0876Calculated value: 535.0877(C₂₇H₁₉O₁₂)

[0205] UV-VISIBLE Absorption Spectrum λ_(max) (in methanol) nm(ε): 207(47,600), 243 (41,800), 314 (34,000), 369 (23,000)

[0206] Infrared Absorption Spectrum υ_(max) (KBr) cm⁻¹: 3444, 1702,1614, 1474, 1289

[0207]¹H-NMR (DMSO-d₆) δ ppm: 2.29, 2.31, 2.67, 2.70, 4.60, 4.65, 4.69,5.97, 6.32, 6.35, 6.89, 6.90, 7.03, 7.17, 7.94, 8.50, 12.50, 9.20-10.80

[0208]¹³C-NMR (DMSO-d₆) δ ppm: 16.6, 17.1, 32.3, 32.4, 56.2, 65.9, 68.2,102.3, 103.0, 103.2, 103.9, 109.9, 110.0, 110.4, 110.5, 111.9, 112.2,118.2, 118.6, 120.5, 125.7, 127.7, 130.0, 131.9, 132.4, 134.8, 138.2,139.1, 140.9, 141.1, 141.5, 150.2, 150.3, 151.7, 152.2, 154.1, 154.6,154.9, 155.8, 156.6, 167.5, 172.6, 172.7, 183.5, 187.2, 199.3, 202.7,202.9

[0209] Solubility: Insoluble: water, hexane Soluble: methanol, DMSO

[0210] Taken together, the structure of SPF-3059-9 was determined as thefollowing formula [24] (tautomer).

[0211] (SPF-3059-12)

[0212] Appearance: Cream-colored powder

[0213] Molecular weight: 560

[0214] Molecular formula: C₂₈H₁₆O₁₃

[0215] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 561(M+H)⁺

[0216] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 559(M−H)⁻

[0217] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M+H)⁺: Measured value: 561.0680Calculated value: 561.0670(C₂₈H₁₇O₁₃)

[0218] UV-VISIBLE Absorption Spectrum λ_(max) (in methanol) nm(ε): 232(37,400), 250sh (34,800), 285 (28,000), 308sh (23,200), 360sh (9,000)

[0219] Infrared Absorption Spectrum υ_(max) (KBr) cm⁻¹: 3080, 1698,1608, 1468, 1291

[0220]¹H-NMR (DMSO-d₆) δ ppm: 2.54 (3H, s), 2.55 (3H, s), 6.82 (1H, d,2.1), 6.87 (1H, s), 6.95 (1H, d, 2.1), 8.22 (1H, s), 8.55 (1H, s),9.50-13.50 (5H, brs)

[0221]¹³C-NMR (DMSO-d₆) δ ppm: 29.1, 32.2, 102.1, 103.0, 109.4, 112.1,113.5, 119.8, 120.0, 121.7, 126.6, 132.0, 133.3, 135.9, 136.7, 141.7,150.6, 152.1, 153.0, 155.4, 157.6, 162.4, 167.4, 167.6, 172.2, 172.9,199.1, 201.1

[0222] Solubility: Insoluble: water, hexane Soluble: methanol, DMSO

[0223] Taken together, the structure of SPF-3059-12 was determined asthe following formula [25].

[0224] (SPF-3059-24)

[0225] Appearance: Cream-colored powder

[0226] Molecular weight: 532

[0227] Molecular formula: C₂₇H₁₆O₁₂

[0228] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 533(M+H)⁺

[0229] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 531(M−H)⁻

[0230] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M+H)⁺: Measured value: 531.0621Calculated value: 531.0564(C₂₇H₁₇O₁₂)

[0231] UV-visible Absorption Spectrum λ_(max) (in methanol) nm(ε): 212(36,900), 229sh(34,500), 283 (26,300), 323 (21,700)

[0232] Infrared Absorption Spectrum υ_(max) (KBr) cm⁻¹: 3447, 1697,1629, 1578, 1470, 1290

[0233]¹H-NMR (DMSO-d₆) δ ppm: 2.52 (3H, s), 2.54 (3H, s), 6.92 (1H, s),6.93 (1H, s), 7.28 (1H, s), 8.13 (1H, s), 8.54 (1H, s), 9.50-13.00 (5H,brs)

[0234]¹³C-NMR (DMSO-d₆) δ ppm: 29.1, 32.3, 102.3, 102.9, 107.9, 110.0,115.8, 119.8, 120.4, 120.7, 126.5, 133.0, 133.3, 136.0, 141.2, 145.0,150.4, 151.1, 152.2, 152.9, 153.0, 154.3, 167.5, 172.6, 173.6, 199.1,201.1

[0235] Solubility: Insoluble: water, hexane Soluble: methanol, DMSO

[0236] Taken together, the structure of SPF-3059-24 was determined asthe following formula [26].

[0237] (SPF-3059-25)

[0238] Appearance: Cream-colored powder

[0239] Molecular formula: C₂₇H₁₆O₁₁

[0240] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 517(M+H)⁺

[0241] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 515(M−H)⁻

[0242] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M+H)⁺: Measured value: 517.0778Calculated value: 517.0771(C₂₇H₁₇O₁₁)

[0243] UV-VISIBLE Absorption Spectrum λ_(max) (in methanol) nm(ε): 215(35,000), 253 (35,100), 276sh(25,200), 323 (23,400)

[0244] Infrared Absorption Spectrum λ_(max) (KBr) cm⁻¹: 3417, 1691,1625, 1471, 1293

[0245]¹H-NMR (DMSO-d₆) δ ppm: 2.54 (6H, s), 6.82 (1H, brs), 6.92 (2H,brs), 7.27 (1H, s), 8.14 (1H, s), 8.53 (1H, s), 9.5-14.0 (4H, brs)

[0246]¹³C NMR (DMSO-d₆) δ ppm: 29.2, 32.3, 102.9, 103.0, 107.8, 109.9,113.0, 115.7, 120.4, 120.6, 126.4, 133.3, 133.4, 136.4 (2C), 145.0,151.2, 152.3, 152.98, 153.01, 157.3, 164.2, 169.4, 172.6, 173.6, 199.2,201.0

[0247] Solubility: Insoluble: water, hexane Soluble: methanol, DMSO

[0248] Taken together, the structure of SPF-3059-25 was determined asthe following formula [27].

[0249] (SPF-3059-26)

[0250] Appearance: Cream-colored powder

[0251] Molecular weight: 488

[0252] Molecular formula: C₂₆H₁₆O₁₀

[0253] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 489(M+H)⁺

[0254] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 487(M−H)⁻

[0255] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M+H)⁺: Measured value: 489.0823Calculated value: 489.0822(C₂₆H₁₇O₁₀₎

[0256] UV-VISIBLE Absorption Spectrum A max (in methanol) nm(ε): 212(31,500), 235 (30,900), 284 (23,900), 324 (19,500)

[0257] Infrared Absorption Spectrum υ_(max) (KBr) cm⁻¹: 3454, 1694,1625, 1517, 1471, 1293

[0258]¹H-NMR (DMSO-d₆) δ ppm: 2.53 (3H, s), 2.54 (3H, s), 6.91 (1H, s),6.92 (1H, s), 7.27 (1H, s), 7.47 (1H, s), 8.11 (1H, s), 8.57 (1H, s)

[0259]¹³C-NMR (DMSO-d₆) δ ppm: 29.1, 32.2, 102.9, 103.0, 107.9, 108.5,113.3, 115.7, 119.8, 120.7, 126.3, 132.7, 133.5, 135.8, 144.6, 145.0,150.8, 151.1, 152.5, 152.9 (2C), 154.7, 173.3, 173.6, 199.1, 201.2

[0260] Solubility: Insoluble: water, hexane Soluble: methanol, DMSO

[0261] Taken together, the structure of SPF-3059-26 was determined asthe following formula [28].

[0262] (SPF-3059-27)

[0263] Appearance: Yellow powder

[0264] Molecular weight: 642

[0265] Molecular formula: C₃₃H₂₂O₁₄

[0266] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 643(M+H)⁺

[0267] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 641(M−H)⁻

[0268] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M+H)⁺: Measured value: 643.1088Calculated value: 643.1089(C₃₃H₂₃O₁₄)

[0269] UV-VISIBLE Absorption Spectrum. λ_(max) (in methanol) nm(ε): 213(46,100), 246 (46,600), 287 (31,700), 354 (19,900)

[0270] Infrared Absorption Spectrum υ_(max) (KBr) cm⁻¹: 3400, 1694,1640, 1604, 1468, 1290

[0271]¹H-NMR (DMSO-d₆) δ ppm:

[0272] 2.41 (3H, s), 2.45 (3H, s), 2.67 (3H, s), 6.34 (1H, s), 6.63 (1H,s), 6.90 (1H, s), 7.48 (1H, d, 2.1), 7.97 (1H, d, 2.1), 8.49 (1H, s),11.9 (1H, brs), 12.5 (1H, brs)

[0273]¹³C-NMR (DMSO-d₆) δ ppm: 27.4, 30.2, 32.7, 102.3, 102.8, 109.8,111.8, 117.4, 119.6, 119.7, 120.3, 126.7, 127.5, 128.4, 133.0, 133.4,135.1, 135.8, 138.6, 139.9, 141.3, 150.5, 151.7, 154.6, 155.8, 157.5,158.3, 167.5, 172.5, 196.3, 200.0, 201.6, 205.3

[0274] Solubility: Insoluble: water, hexane Soluble: methanol, DMSO

[0275] Taken together, the structure of SPF-3059-27 was determined asthe following formula [29].

[0276] (SPF-3059-28)

[0277] Appearance: Cream-colored powder

[0278] Molecular weight: 532

[0279] Molecular formula: C₂₇H₁₆O₁₂

[0280] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 533(M+H)⁺

[0281] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 531(M−H)⁻

[0282] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M+H)⁺: Measured value: 533.0735Calculated value: 533.0721(C₂₇H₁₇O₁₂)

[0283] UV-VISIBLE Absorption Spectrum λ_(max) (in methanol) nm(ε): 217(35,300), 236 (34,100), 309 (26,100)

[0284] Infrared Absorption Spectrum υ_(max) (KBr) cm⁻¹: 3502, 3096,1690, 1598, 1503, 1434, 1303

[0285]¹H-NMR (DMSO-d₆) δ ppm: 2.39 (3H, s), 2.71 (3H, s), 6.52 (1H, s),6.85 (1H, d, 2.1), 6.92 (1H, d, 2.1), 6.98 (1H, d, 2.1), 8.25 (1H, s),9.5-13.5 (5H, brs)

[0286]¹³C-NMR (DMSO-d₆) δ ppm: 17.3, 32.4, 102.3, 103.3, 110.0, 112.1,112.3, 113.4, 118.8, 120.6, 127.6, 128.9, 132.1, 136.1, 138.8, 140.9,150.2, 152.0, 154.1, 157.8, 162.2, 162.6, 167.5, 169.2, 172.6, 175.3,202.7

[0287] Solubility: Insoluble: water, hexane Soluble: methanol, DMSO

[0288] Taken together, the structure of SPF-3059-28 was determined asthe following formula [30].

[0289] (SPF-3059-29)

[0290] Appearance: Cream-colored powder

[0291] Molecular weight: 548

[0292] Molecular formula: C₂₈H₂₀O₁₂

[0293] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 549(M+H)⁺

[0294] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 547(M−H)⁻

[0295] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M+H)⁺: Measured value: 549.1027Calculated value: 549.1034(C₂₈H₂₁O₁₂)

[0296] UV-VISIBLE Absorption Spectrum λ_(max) (in methanol) nm(ε): 226(48,900), 316 (26,200), 352sh (17,400) Infrared Absorption Spectrumυ_(max) (KBr) cm⁻¹: 3388, 1687, 1662, 1626, 1469, 1296

[0297]¹H-NMR (DMSO-d₆) δ ppm:

[0298] 2.23 (3H, s), 2.72 (3H, s), 3.11 (3H, s), 3.98 (2H, brs), 6.89(1H, s), 6.93 (1H, s), 7.44 (1H, s), 8.27 (1Hs), 9.00-13.00 (5H, brs)

[0299]¹³C-NMR (DMSO-d₆) δ ppm: 16.8, 32.4, 57.8, 64.4, 102.3, 103.1,108.6, 112.0, 113.5, 118.46, 118.48, 119.1, 127.7, 128.1, 131.8, 138.9,141.8, 144.3, 150.6, 150.7, 151.9, 152.6, 154.2, 162.1, 167.8, 173.5,174.6, 202.7

[0300] Solubility: Insoluble: water, hexane Soluble: methanol, DMSO

[0301] Taken together, the structure of SPF-3059-29 was determined asthe following formula [31].

[0302] (SPF-3059-30)

[0303] Appearance: Cream-colored powder

[0304] Molecular weight: 490

[0305] Molecular formula: C₂₆H₁₈O₁₀

[0306] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 491(M+H)⁺

[0307] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 489(M−H)⁻

[0308] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M+H)⁺: Measured value: 491.0966Calculated value: 491.0979(C₂₆H₁₉O₁₀)

[0309] UV-VISIBLE Absorption Spectrum Amax (in methanol) nm(ε): 206(36,000), 240 (32,700), 315 (26,500), 372 (17,900)

[0310] Infrared Absorption Spectrum λ_(max) (KBr) cm⁻¹: 3396, 1704,1618, 1518, 1479, 1294

[0311]¹H-NMR (DMSO-d₆) δ ppm: 2.28, 2.30, 2.67, 2.69, 4.62, 4.66, 4.70,4.96, 6.32, 6.36, 6.90, 6.91, 7.03, 7.17, 7.43, 7.98, 8.54, 9.20-10.80

[0312]¹³C-NMR (DMSO-d₆) δ ppm: 16.5, 17.0, 32.3, 32.4, 56.2, 65.9, 68.3,103.0, 103.2, 103.8, 108.6, 110.4, 111.8, 113.4, 118.6, 125.6, 127.5,129.5, 132.1, 132.6, 134.4, 137.9, 138.8, 141.2, 141.5, 144.3, 150.6,152.0, 152.5, 154.3, 154.6, 154.9, 155.8, 156.6, 172.6, 173.4, 183.5,187.2, 199.3, 202.7, 202.9

[0313] Solubility: Insoluble: water, hexane Soluble: methanol, DMSO

[0314] Taken together, the structure of SPF-3059-30 was determined asthe following formula [32] (tautomer).

[0315] (SPF-3059-34)

[0316] Appearance: Yellow powder

[0317] Molecular weight: 550

[0318] Molecular formula: C₂₇H₁₈O₁₃

[0319] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 551(M+H)⁺

[0320] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 549(M−H)⁻

[0321] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M+H)⁺: Measured value: 551.0846Calculated value: 551.0826(C₂₇H₁₉O₁₃)

[0322] UV-VISIBLE Absorption Spectrum A max (in methanol) nm(ε): 211(35,600), 240 (31,100), 283 (24,100), 349 (14,500)

[0323]¹H-NMR (DMSO-d₆) δ ppm: 2.18 (3H, s), 2.66 (3H, s), 4.39 (1H, d,11.9), 4.64 (1H, d, 11.9), 6.37 (1H, s), 6.84 (1H, s), 7.09 (1H, s),8.47 (1H, brs)

[0324]¹³C-NMR (DMSO-d₆) δ ppm: 16.8, 32.4, 72.5, 80.0, 102.1, 103.0,109.5, 110.6, 111.1, 117.7, 120.0, 126.5, 131.8, 133.6, 138.6, 141.4,141.5, 150.5, 151.7, 154.9, 155.0, 156.2, 167.7, 172.6, 188.5, 202.8,204.0

[0325] Solubility: Insoluble: water, hexane Soluble: methanol, DMSO

[0326] Taken together, the structure of SPF-3059-34 was determined asthe following formula [33].

[0327] (SPF-3059-35)

[0328] Appearance: Cream-colored powder

[0329] Molecular weight: 546

[0330] Molecular formula: C₂₈H₁₈O₁₂

[0331] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 547(M+H)⁺

[0332] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 545(M−H)⁻

[0333] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M+H)⁺: Measured value: 547.0911Calculated value: 547.0877(C₂₈H₁₉O₁₂)

[0334] UV-VISIBLE Absorption Spectrum λ_(max) (in methanol) nm(ε): 216(38,600), 237 (37,300), 307 (30,100), 356sh (8,800)

[0335] Infrared Absorption Spectrum υ_(max) (KBr) cm⁻¹: 3460, 3076,1734, 1699, 1629, 1466, 1302

[0336]¹H-NMR (DMSO-d₆) δ ppm: 2.39 (3H, s), 2.71 (3H, s), 3.82 (3H, s),6.49 (1H, s), 6.86 (1H, d, 2.1), 6.92 (1H, s), 7.01 (1H, d, 2.1), 8.25(1H, s), 9.5-13.5 (4H, brs)

[0337]¹³C-NMR (DMSO-d₆) δ ppm: 17.3, 32.4, 52.5, 102.3, 103.8, 110.0,112.1, 112.8, 113.5, 118.8, 120.6, 127.7, 129.0, 132.1, 134.3, 138.8,140.9, 150.3, 152.1, 154.1, 157.7, 162.2, 162.8, 167.5, 168.6, 172.6,175.1, 202.7

[0338] Solubility: Insoluble: water, hexane Soluble: methanol, DMSO

[0339] Taken together, the structure of SPF-3059-35 was determined asthe following formula [34].

[0340] (SPF-3059-36)

[0341] Appearance: Cream-colored powder

[0342] Molecular weight: 598

[0343] Molecular formula: C₃₂H₂₂O₁₂

[0344] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 599(M+H)⁺

[0345] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 597(M−H)⁻

[0346] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M+H)⁺: Measured value: 599.1198Calculated value: 599.1190(C₃₂H₂₃O₁₂)

[0347] UV-VISIBLE Absorption Spectrum Amax (in methanol) nm(ε): 213(46,300), 246 (48,700), 287 (33,200), 360 (20,000)

[0348]¹H-NMR (DMSO-d₆) δ ppm:

[0349] 2.41 (3H, s), 2.45 (3H, s), 2.66 (3H, s), 6.34 (1H, s), 6.63 (1H,s), 6.90 (1H, s), 7.46 (1H, d, 2.0), 7.46 (1H, s), 7.95 (1H, d, 2.0),8.52 (1H, s)

[0350]¹³C-NMR (DMSO-d₆) δ ppm: 27.3, 30.2, 32.7, 102.8, 103.1, 108.6,111.7, 113.4, 117.3, 119.6 (2C), 126.7, 127.5, 128.5, 133.2, 133.4,135.1, 135.6, 138.6, 139.7, 144.6, 150.8, 152.1, 154.6, 155.9, 157.5,158.4, 173.3, 196.3, 200.0, 201.6, 205.2

[0351] Solubility: Insoluble: water, hexane Soluble: methanol, DMSO

[0352] Taken together, the structure of SPF-3059-36 was determined asthe following formula [35].

[0353] (SPF-3059-37)

[0354] Appearance: Yellow powder

[0355] Molecular weight: 806

[0356] Molecular formula: C₄₁H₂₆O₁₈

[0357] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 807(M+H)⁺

[0358] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 805(M−H)⁻

[0359] High Resolution Fast Atom Bombardment Mass Spectrum (HRFAB-MS)m/z(M−H)⁻: Measured value: 807.1197Calculated value: 807.1198(C₄₁H₂₇O₁₈)

[0360] UV-VISIBLE Absorption Spectrum λ_(max) (in methanol) nm(ε): 219(68,900), 323 (30,700), 349 (30,600)

[0361]¹H-NMR (DMSO-d₆) δ ppm: 2.08 (3H, s), 2.20 (3H, s), 2.53 (3H, s),3.42 (1H, d, 15.6), 3.56 (1H, d, 15.6), 4.64 (1H, d, 13.4), 4.71 (1H, d,13.4), 6.27 (1H, s), 6.83 (1H, s), 6.84 (1H, s), 6.88 (1H, s), 8.15 (1H,s), 9.0-13.0 (8H, brs)

[0362]¹³C-NMR (DMSO-d₆) δ ppm: 16.6, 17.7, 20.0, 32.0, 62.3, 102.1,102.25, 103.8, 106.0, 108.4, 108.7, 109.9, 111.55, 118.56, 119.2, 119.6,120.4, 121.7, 127.9, 129.13, 131.1, 139.5, 140.6, 141.00, 141.7, 150.1,150.23, 150.46, 150.51, 151.6, 152.3, 154.09, 154.17, 158.7, 161.1,167.7, 168.0, 172.5, 173.2, 175.1, 202.2

[0363] Solubility: Insoluble: water, hexane Soluble: methanol, DMSO

[0364] Taken together, the structure of SPF-3059-37 was determined asthe following formula [36].

[0365] (SPF-3059-39)

[0366] Appearance: Yellow powder

[0367] Molecular weight: 548

[0368] Molecular formula: C₂₇H₁₆O₁₃

[0369] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(positive): 549(M+H)⁺

[0370] Fast Atom Bombardment Mass Spectrum (FAB-MS) m/z(negative): 547(M−H)⁻

[0371] UV-VISIBLE Absorption Spectrum λ_(max) (in methanol) nm(ε): 223(40,000), 319 (23,900), 349 (20,100)

[0372]¹H-NMR (DMSO-d₆) δ ppm: 2.37 (3H, s), 2.71 (3H, s), 6.47 (3H, s),6.91 (1H, s), 6.98 (1H, s), 8.22 (1H, s), 9.0-13.0 (6H, brs)

[0373]¹³C-NMR (DMSO-d₆) δ ppm: 17.3, 32.4, 102.3, 102.7, 109.9, 111.49,112.4, 118.64, 118.8, 120.5, 127.5, 129.12, 132.1, 138.8, 140.96, 142.5,150.29, 151.1, 152.0, 152.6, 154.24, 162.1, 167.5, 167.9, 172.6, 175.5,202.7

[0374] Solubility: Insoluble: water, hexane Soluble: methanol, DMSO

[0375] Taken together, the structure of SPF-3059-39 was determined asthe following formula [37].

EXAMPLE 15 Inhibitory Action of the Novel Compounds of the PresentInvention to the Collapse Activity of Sema 3A

[0376] In a similar manner as in Example 2, inhibitory actions of thenovel compounds of the present invention to the collapse activity ofSema 3A were measured. IC50 were as follows and all of them stronglyinhibited Sema 3A. Compound IC50 (μg/ml) SPF-3059-3 0.2 SPF-3059-4 2.0SPF-3059-6 0.1 SPF-3059-7 1.0 SPF-3059-9 0.1 SPF-3059-12 2.0 SPF-3059-240.1 SPF-3059-25 4.0 SPF-3059-26 0.2 SPF-3059-27 0.5 SPF-3059-28 2.0SPF-3059-29 0.1 SPF-3059-30 0.2 SPF-3059-34 0.1 SPF-3059-35 2.0SPF-3059-36 4.0 SPF-3059-37 <0.1 SPF-3059-39 0.1

EXAMPLE 16 Inhibition of the Neurite Outgrowth-Inhibitory Action ofSema3A by the Inhibitor

[0377] In a similar manner as in Example 5, the inhibitory actions ofthe novel compounds of the present invention to the neuriteoutgrowth-inhibitory action of Sema3A were measured. The results of themeasurement are presented below. Compound concentration (μg/ml) Compound2 6 20 SPF-3059-2 +++ NT NT SPF-3059-3 +++ NT NT SPF-3059-5 ++ NT NTSPF-3059-4 − + ++ SPF-3059-6 ++ ++ +++ SPF-3059-7 + + ++ SPF-3059-12 + +++ PBS (Control) − − −

[0378] This result demonstrates that the neurite outgrowth-inhibitoryaction presented by Sema 3A can be persistently inhibited by the novelcompounds of the present invention.

EXAMPLE 17 Determination of Cytotoxicity of the Novel Compounds of thePresent Invention

[0379] The effects of the compounds to cell proliferation weredetermined by using COS7 cells, in a similar manner as in Example 10.The determination results of the cell proliferation inhibition of thenovel compounds of the present invention were as follows. It wasrevealed that no cytotoxicity was observed at a concentration as high as50 to 1000 folds of the concentration where semaphorin activities can beobserved. Subject Compound IC50 (μg/ml) SPF-3059-3 >100 SPF-3059-4 >100SPF-3059-6 >100 SPF-3059-7 >100 SPF-3059-9 >100 SPF-3059-12 >100SPF-3059-24 >100 SPF-3059-29 >100 SPF-3059-30 >100

EXAMPLE 18 Production of Salts of the Compounds of the Present Invention

[0380] A compound of the present invention is dissolved in methanol toprepare 1 mM solution. Methanol solution of 1 mM sodium hydroxide isadded to 1 ml of the above solution by 2 ml when a compound of thepresent invention has two carboxyl groups and by 1 ml when it has onecarboxyl group, and is mixed thoroughly. The solvent of the solution isevaporated under reduced pressure and the residues are dried, and thus 1μmol of sodium salt of a compound of the present invention is obtained.

INDUSTRIAL APPLICABILITY

[0381] The semaphorin inhibitors as an active ingredient for nerveregeneration promoters of the present invention have the peripheral orcentral nerve regeneration promoting action even at a low concentrationand have suppressing action on the growth cone collapse activity ofsemaphorin and/or on the neurite outgrowth inhibitory activity ofsemaphorin in a collagen gel. Especially, a low molecular weightcompound semaphorin inhibitor with a molecular weight of 1000 or less isadvantageously used as a preventive or a remedy for various neuropathicand neurodegenerative diseases because of its significant moleculardiffusibility, membrane permeability, organ distribution, blood-brainbarrier permeability in particular, or the like.

1. A nerve regeneration promoter containing an inhibitor for a nerveoutgrowth repelling factor as an active ingredient.
 2. The nerveregeneration promoter according to claim 1, wherein the inhibitor for anerve outgrowth repelling factor is a semaphorin inhibitor.
 3. The nerveregeneration promoter according to claim 2, wherein the semaphorininhibitor is a class 3 semaphorin inhibitor.
 4. The nerve regenerationpromoter according to claim 3, wherein the class 3 semaphorin inhibitoris a semaphorin 3A inhibitor.
 5. The nerve regeneration promoteraccording to claim 2, wherein the semaphorin inhibitor is a class 6semaphorin inhibitor.
 6. The nerve regeneration promoter according toclaim 5, wherein the class 6 semaphorin inhibitor is a semaphorin 6Cinhibitor.
 7. The nerve regeneration promoter according to any of claims2-6, wherein the semaphorin inhibitor is a compound having promotingaction on the central and/or the peripheral nerve regeneration.
 8. Thenerve regeneration promoter according to any of claims 2-7, wherein thesemaphorin inhibitor is a compound having suppressing action on thegrowth cone collapse activity of semaphorin and/or on the nerveoutgrowth inhibitory activity of semaphorin in a collagen gel.
 9. Thenerve regeneration promoter according to claim 8, wherein the compoundhaving suppressing activity for the growth cone collapse activity and/orsuppressing action on the nerve outgrowth inhibitory activity in acollagen gel exhibits said suppressing action at a concentration of 100μg/ml or below.
 10. The nerve regeneration promoter according to claim9, wherein the compound having suppressing activity for the growth conecollapse activity and/or suppressing action on the nerve outgrowthinhibitory activity in a collagen gel exhibits said suppressing actionat a concentration of 10 μg/ml or below.
 11. The nerve regenerationpromoter according to claim 10, wherein the compound having suppressingactivity on the growth cone collapse activity and/or suppressing actionon the nerve outgrowth inhibitory activity in a collagen gel exhibitssaid suppressing action at a concentration of 3 μg/ml or below.
 12. Thenerve regeneration promoter according to any of claims 2-11, wherein thesemaphorin inhibitor is a compound which inhibits the function ofsemaphorin by contacting said semaphorin.
 13. The nerve regenerationpromoter according to any of claims 2-12, wherein the semaphorininhibitor is a compound which does not substantially affect cellproliferation.
 14. The nerve regeneration promoter according to any ofclaims 2-13, wherein the semaphorin inhibitor is a compound with amolecular weight of 1000 or less.
 15. The nerve regeneration promoteraccording to any of claims 2-14, wherein the semaphorin inhibitor is anon-peptide and a non-nucleotide compound.
 16. The nerve regenerationpromoter according to claim 15, wherein the non-peptide and thenon-nucleotide compound is obtained from cultivating Penicillium sp.SPF-3059.
 17. The nerve regeneration promoter according to claim 16,wherein the non-peptide and the non-nucleotide compound obtained fromcultivating Penicillium sp. SPF-3059 contains a group represented byformulae [1], [2], [4] or [5] and/or a group represented by formulae [6]or [7] in the molecule,

represents a single bond or a double bond, R¹ represents a hydrogenatom, a carboxyl group or an alkoxycarbonyl group and R² represents ahydrogen atom, a hydroxyl group or an acyloxy group,

wherein R¹ and R² have the same meanings as in formula [1] and R³represents a hydrogen atom, a methoxymethyl group or formula [3],

wherein R¹ and R² have the same meanings as in formula [1],

wherein R⁴ represents a hydrogen atom, a carboxyl group or analkoxycarbonyl group and R⁵ represents a hydrogen atom, a hydroxyl groupor an acyloxy group,

wherein R⁴ and R⁵ have the same meanings as in formula [6].
 18. Asemaphorin inhibitor containing a compound which has a group representedby formulae [1], [2], [4] or [5] and a group represented by formulae [6]or [7] in the molecule, a pharmaceutically acceptable salt thereof or aderivative thereof, as an active ingredient,

represents a single bond or a double bond, R¹ represents a hydrogenatom, a carboxyl group or an alkoxycarbonyl group and R² represents ahydrogen atom, a hydroxyl group or an acyloxy group,

wherein R¹ and R² have the same meanings as in formula [1] and R³represents a hydrogen atom, a methoxymethyl group or formula [3],

wherein R¹ and R² have the same meanings as in formula [1],

wherein R⁴ represents a hydrogen atom, a carboxyl group or analkoxycarbonyl group and R⁵ represents a hydrogen atom, a hydroxyl groupor an acyloxy group,

wherein R⁴ and R⁵ have the same meanings as in formula [6].
 19. Asemaphorin inhibitor containing a compound represented by formula [8], apharmaceutically acceptable salt thereof or a derivative thereof, as anactive ingredient,

wherein R⁴ and R⁵ have the same meanings as in formula [6] and R⁶ and R⁷are represented by either (1) or (2) below: (1) R⁶ represents a methylgroup and R⁷ represents a group shown by formulae [2], [9] or [10],

wherein R¹ and R² have the same meanings as in formula [1] and R³represents a hydrogen atom, a methoxymethyl group or formula [3],

wherein R¹ and R² have the same meanings as in formula [1],

wherein R¹ and R² have the same meanings as in formula [1], (2) R⁶represents a group shown by formulae [5] or [11] and R⁷ represents anacetyl group.


20. A semaphorin inhibitor containing a compound represented by formula[12], a pharmaceutically acceptable salt thereof or a derivativethereof, as an active ingredient,

wherein R¹, R², R⁴ and R⁵ have the same meanings as in formulae [1] and[6].
 21. The semaphorin inhibitor according to claim 20 which contains acompound wherein at least one of R² and R⁵ represents a hydroxyl groupin formula [12], a pharmaceutically acceptable salt thereof or aderivative thereof, as an active ingredient.
 22. The semaphorininhibitor according to claim 21 which contains a compound wherein R²represents a hydroxyl group in formula [12], a pharmaceuticallyacceptable salt thereof or a derivative thereof, as an activeingredient.
 23. The semaphorin inhibitor according to claim 21 whichcontains a compound wherein R² and R⁵ represent a hydroxyl group informula [12], a pharmaceutically acceptable salt thereof or a derivativethereof, as an active ingredient.
 24. The semaphorin inhibitor accordingto any of claims 20-23 which contains a compound wherein R⁴ represents acarboxyl group in formula [12], a pharmaceutically acceptable saltthereof or a derivative thereof, as an active ingredient.
 25. Thesemaphorin inhibitor according to claim 20 which contains a compoundwherein R¹ and R⁴ represent a carboxyl group and R² represents ahydroxyl group in formula [12], a pharmaceutically acceptable saltthereof or a derivative thereof, as an active ingredient.
 26. A nerveregeneration promoter containing the semaphorin inhibitor according toany of claims 18-25 as an active ingredient.
 27. A compound representedby formula [12], wherein at least one of R¹, R², R⁴ and R⁵ isrepresented by a hydrogen atom, a pharmaceutically acceptable saltthereof or a derivative thereof,

wherein R¹, R², R⁴ and R⁵ have the same meanings as in formulae [1] and[6].
 28. The compound according to claim 27 wherein at least one of R²and R⁵ represents a hydroxyl group, a pharmaceutically acceptable saltthereof or a derivative thereof.
 29. The compound according to claim 28wherein R² represents a hydroxyl group, a pharmaceutically acceptablesalt thereof or a derivative thereof.
 30. The compound according toclaim 28 wherein R² and R⁵ represent a hydroxyl group, apharmaceutically acceptable salt thereof or a derivative thereof. 31.The compound according to any of claims 27-30 wherein R⁴ represents acarboxyl group, a pharmaceutically acceptable salt thereof or aderivative thereof.
 32. The compound according to claim 27 wherein R¹and R⁴ represent a carboxyl group and R² represents a hydroxyl group, apharmaceutically acceptable salt thereof or a derivative thereof.
 33. Asemaphorin inhibitor containing a compound represented by formula [13],a pharmaceutically acceptable salt thereof or a derivative thereof, asan active ingredient,

wherein R¹, R², R⁴ and R⁵ have the same meanings as in formulae [1] and[6].
 34. The semaphorin inhibitor according to claim 33 which contains acompound wherein at least one of R² and R⁵ represents a hydroxyl groupin formula [13], a pharmaceutically acceptable salt thereof or aderivative thereof, as an active ingredient.
 35. The semaphorininhibitor according to claim 34 which contains a compound wherein R²represents a hydroxyl group in formula [13], a pharmaceuticallyacceptable salt thereof or a derivative thereof, as an activeingredient.
 36. The semaphorin inhibitor according to claim 34 whichcontains a compound wherein R² and R⁵ represent a hydroxyl group informula [13], a pharmaceutically acceptable salt thereof or a derivativethereof, as an active ingredient.
 37. The semaphorin inhibitor accordingto any of claims 33-36 which contains a compound wherein R⁴ represents acarboxyl group in formula [13], a pharmaceutically acceptable saltthereof or a derivative thereof, as an active ingredient.
 38. Thesemaphorin inhibitor according to claim 33 which contains a compoundwherein R² and R⁵ represent a hydroxyl group, R¹ represents a carboxylgroup and R⁴ represents a hydrogen atom in formula [13], apharmaceutically acceptable salt thereof or a derivative thereof, as anactive ingredient.
 39. The semaphorin inhibitor according to claim 33which contains a compound wherein R¹ and R⁴ represent a carboxyl groupand R⁵ represents a hydroxyl group in formula [13], a pharmaceuticallyacceptable salt thereof or a derivative thereof, as an activeingredient.
 40. A nerve regeneration promoter containing the semaphorininhibitor according to any of claims 33-39 as an active ingredient. 41.A compound represented by formula [13], wherein at least one of R¹, R²and R⁵ represents a hydrogen atom, a pharmaceutically acceptable saltthereof or a derivative thereof,

wherein R¹, R², R⁴ and R⁵ have the same meanings as in formulae [1] and[6].
 42. The compound according to claim 41 wherein at least one of R²and R⁵ represents a hydroxyl group, a pharmaceutically acceptable saltthereof or a derivative thereof.
 43. The compound according to claim 42wherein R² represents a hydroxyl group, a pharmaceutically acceptablesalt thereof or a derivative thereof.
 44. The compound according toclaim 42 wherein R² and R⁵ represent a hydroxyl group, apharmaceutically acceptable salt thereof or a derivative thereof. 45.The compound according to any of claims 41-44 wherein R⁴ represents acarboxyl group, a pharmaceutically acceptable salt thereof or aderivative thereof.
 46. The compound according to claim 41 wherein R²represents a hydroxyl group and R⁴ represents a carboxyl group, apharmaceutically acceptable salt thereof or a derivative thereof. 47.The compound according to claim 41 wherein R¹ and R⁴ represent acarboxyl group and R⁵ represents a hydroxyl group, a pharmaceuticallyacceptable salt thereof or a derivative thereof.
 48. A compoundrepresented by formula [14], a pharmaceutically acceptable salt thereofor a derivative thereof,

wherein R¹, R², R³, R⁴ and R⁵ have the same meanings as in formulae [2]and [6].
 49. The compound according to claim 48 wherein at least one ofR² and R⁵ represents a hydroxyl group, a pharmaceutically acceptablesalt thereof or a derivative thereof.
 50. The compound according toclaim 49 wherein R² represents a hydroxyl group, a pharmaceuticallyacceptable salt thereof or a derivative thereof.
 51. The compoundaccording to claim 49 wherein R² and R⁵ represent a hydroxyl group, apharmaceutically acceptable salt thereof or a derivative thereof. 52.The compound according to any of claims 48-51 wherein R⁴ represents acarboxyl group, a pharmaceutically acceptable salt thereof or aderivative thereof.
 53. The compound according to claim 48 wherein R¹and R⁴ represent a carboxyl group and R² and R⁵ represent a hydroxylgroup, a pharmaceutically acceptable salt thereof or a derivativethereof.
 54. The compound according to claim 48 wherein R¹ represents acarboxyl group, R² and R⁵ represent a hydroxyl group and R³ represents amethoxymethyl group, a pharmaceutically acceptable salt thereof or aderivative thereof.
 55. The compound according to claim 48 wherein R¹represents a carboxyl group or a methoxycarbonyl group, R⁴ represents acarboxyl group, R³ represents a hydrogen atom and R⁵ represents ahydroxyl group, a pharmaceutically acceptable salt thereof or aderivative thereof.
 56. The compound according to claim 55 wherein R¹represents a carboxyl group or a methoxycarbonyl group, R⁴ represents acarboxyl group, R² and R³ represent a hydrogen atom and R⁵ represents ahydroxyl group, a pharmaceutically acceptable salt thereof or aderivative thereof.
 57. The compound according to any of claims 48-53,wherein R³ represents a group shown by formula [3], a pharmaceuticallyacceptable salt thereof or a derivative thereof.


58. A semaphorin inhibitor containing a compound of any of claims 48-57,a pharmaceutically acceptable salt thereof or a derivative thereof, asan active ingredient.
 59. A nerve regeneration promoter containing thesemaphorin inhibitor according to claim 58 as an active ingredient. 60.A compound represented by formula [15], a pharmaceutically acceptablesalt thereof or a derivative thereof,

wherein R⁴ and R⁵ have the same meanings as in formula [6].
 61. Thecompound according to claim 60 wherein R⁵ represents a hydroxyl group, apharmaceutically acceptable salt thereof or a derivative thereof. 62.The compound according to claim 60 or 61 wherein R⁴ represents acarboxyl group, a pharmaceutically acceptable salt thereof or aderivative thereof.
 63. A semaphorin inhibitor containing a compound ofany of claims 60-62, a pharmaceutically acceptable salt thereof or aderivative thereof, as an active ingredient.
 64. A nerve regenerationpromoter containing the semaphorin inhibitor of claim 63 as an activeingredient.
 65. A compound represented by formula [16], apharmaceutically acceptable salt thereof or a derivative thereof,

wherein R¹, R², R⁴ and R⁵ have the same meanings as in formulae [1] and[6].
 66. The compound according to claim 65 wherein at least one of R²and R⁵ represents a hydrogen atom, a pharmaceutically acceptable saltthereof or a derivative thereof.
 67. The compound according to claim 65wherein R² and R⁵ represent a hydroxyl group, a pharmaceuticallyacceptable salt thereof or a derivative thereof.
 68. The compoundaccording to any of claims 65-67 wherein R⁴ represents a carboxyl group,a pharmaceutically acceptable salt thereof or a derivative thereof. 69.A semaphorin inhibitor containing a compound of any of claims 65-68, apharmaceutically acceptable salt thereof or a derivative thereof, as anactive ingredient.
 70. A nerve regeneration promoter containing thesemaphorin inhibitor of claim 69 as an active ingredient.
 71. Apreventive or a remedy for neuropathic diseases and/or neurodegenerativediseases containing the nerve regeneration promoter of any of claims1-17, 26, 40, 59, 64 or
 70. 72. The preventive or the remedy forneuropathic diseases and/or neurodegenerative diseases according toclaim 71, wherein said neuropathic diseases and/or neurodegenerativediseases are accompanied with a spinal nerve injury and/or a peripheralnerve injury.
 73. The preventive or the remedy for neuropathic diseasesand/or neurodegenerative diseases according to claim 71, wherein saidneuropathic diseases and/or neurodegenerative diseases are olfactoryabnormality, traumatic neuropathy, cerebral infarctional neuropathy,facial nerve paralysis, diabetic neuropathy, glaucoma, retinitispigmentosa, Alzheimer's disease, Parkinson's disease, neurodegenerativediseases, muscular hypoplastic lateral sclerosis, Lou Gehrig's disease,Huntington's chorea, cerebral infarction or traumatic neurodegenerativediseases.
 74. A process for producing the compound according to any ofclaims 27-32, wherein the process comprises cultivating acompound-producing fungus of any of claims 27-32 which belongs to thegenus Penicillium and collecting said compound from the culture.
 75. Theprocess for producing the compound according to any of claims 41-47,wherein the process comprises cultivating a compound-producing fungus ofany of claims 41-47 which belongs to the genus Penicillium andcollecting said compound from the culture.
 76. The process for producingthe compound according to any of claims 48-57, wherein the processcomprises cultivating a compound-producing fungus of any of claims 48-57which belongs to the genus Penicillium and collecting said compound fromthe culture.
 77. The process for producing the compound according to anyof claims 60-62, wherein the process comprises cultivating acompound-producing fungus of any of claims 60-62 which belongs to thegenus Penicillium and collecting said compound from the culture.
 78. Theprocess for producing the compound according to any of claims 65-68,wherein the process comprises cultivating a compound-producing fungus ofany of claims 65-68 which belongs to the genus Penicillium andcollecting said compound from the culture.
 79. The production processaccording to any of claims 74-78, wherein the producing fungus whichbelongs to the genus Penicillium is Penicillium sp. SPF-3059. 80.Penicillium sp. SPF-3059 (FERM BP-7663) or a fungus strain induced fromsaid SPF-3059.